Long-acting coagulation factors and methods of producing same

ABSTRACT

Polypeptides comprising at least one carboxy-terminal peptide (CTP) of chorionic gonadotropin attached to the carboxy terminus but not to the amino terminus of a coagulation factor and polynucleotides encoding the same are disclosed. Pharmaceutical compositions comprising the polypeptides and polynucleotides of the invention and methods of using and producing same are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 13/932,839, filed on Jul. 1, 2013, which is acontinuation-in-part of U.S. patent application Ser. No. 13/759,860,filed on Feb. 5, 2013, which is a continuation-in-part of U.S. patentapplication Ser. No. 13/372,540, filed Feb. 14, 2012, which is acontinuation-in-part of U.S. patent application Ser. No. 12/826,754,filed Jun. 30, 2010 and now U.S. Pat. No. 8,476,234, which claims thebenefit of U.S. Provisional Application Ser. No. 61/224,366, filed Jul.9, 2009; and is a continuation-in-part application of InternationalApplication No. PCT/IL2013/050107, filed Feb. 5, 2013 all of which arehereby incorporated in their entirety herein.

FIELD OF INVENTION

Polypeptides comprising at least one carboxy-terminal peptide (CTP) ofchorionic gonadotropin attached to the carboxy terminus of a coagulationfactor and polynucleotides encoding the same are disclosed.Pharmaceutical compositions comprising the polypeptides andpolynucleotides of the invention and methods of using and producing sameare also disclosed.

BACKGROUND OF THE INVENTION

The development of coagulation factor replacement therapy hastransformed the lives of many individuals with hemophilia. Hemophilia isa group of hereditary genetic disorders that impair the body's abilityto control blood clotting or coagulation. Patients with hemophilia donot produce adequate amounts of Factor VIII or Factor IX proteins, whichare necessary for effective blood clotting. In severe hemophiliacs evena minor injury can result in blood loss that continues for days orweeks, and complete healing may not occur, leading to the potential fordebilitating permanent damage to joints and other organs, and prematuredeath.

One type of hemophilia, Hemophilia B, is an X-linked bleeding disordercaused by a mutation in the Factor IX (FIX) gene, resulting in adeficiency of the procoagulant activity of FIX. Hemophilia B patientshave spontaneous soft tissue hemorrhages and recurrent hemarthroses thatoften lead to a crippling arthopathy. Current treatment for thesepatients includes an intravenous administration of recombinant FIX.However issues of cost and relatively rapid clearance of FIX from thecirculation make developing a long-acting FIX a challenging task.

Commercial availability of FVIII and FIX has led to improved control oflife-threatening bleedings episodes. Many patients receive prophylactictherapy, which reduces the risk of bleeding and its associatedcomplications. However, a significant proportion of patients (10-30%)develop inhibitory antibodies to exogenously administered FVIII and FIX.Administration of FVIIa, which is a bypassing product, can inducehomeostasis and provide an effective treatment for patients withinhibitory Abs.

Recombinant FVIIa (NovoSeven®) is commercially available and wasapproved in 1996 for treatment of bleeding episodes in hemophiliapatients with inhibitors. However, rFVIIa is rapidly cleared with aterminal half-life of 2.5 hours. As a result, patients generally requiremultiple, frequent infusions (2-3 doses given in 2-3 hour intervals) toachieve adequate homeostasis following a mild to moderate bleed.Consequently, there is much interest in developing a long-acting form ofFVIIa that would prolong the duration of haemostatic activity followinga single dose and allow much less frequent dosing. A long-acting FVIIawould also increase the feasibility of long-term prophylactic therapy.

Various technologies are being developed for prolonging the half-life ofFVIIa. However, there remains a need to achieve a prolonged half-life ofthis protein while preserving its biological activity and ensuring thatthe modifications do not induce significant immunogenicity. The presentinvention addresses this need by attaching gonadotropin carboxy terminalpeptides (CTPs) to FVIIa, thereby modifying it to prolong its half-lifeand biological activity.

SUMMARY OF THE INVENTION

In one embodiment, the present invention relates to a CTP-modifiedpolypeptide consisting of a coagulation factor and three chorionicgonadotropin carboxy terminal peptides (CTPs) attached to the carboxyterminus of said coagulation factor. In one embodiment, the sequence ofat least one CTP is selected from the group consisting of: SEQ ID NO: 1and SEQ ID NO: 2. In one embodiment, at least one CTP is glycosylated.In one embodiment, at least one CTP is truncated. In one embodiment, atleast one CTP is attached to said coagulation factor via a linker. Inone embodiment, the linker is a peptide bond.

In one embodiment, the present invention relates to a CTP-modifiedpolypeptide consisting of a coagulation factor and three chorionicgonadotropin carboxy terminal peptides (CTPs) attached to the carboxyterminus of the coagulation factor, wherein in one embodiment thecoagulation faction is a vitamin K dependent glycoprotein. In anotherembodiment, the coagulation factor is Factor IX. In yet anotherembodiment, the coagulation factor is Factor VII. In still anotherembodiment, the coagulation factor is activated FVII (FVIIa).

In one embodiment, the present invention relates to a pharmaceuticalcomposition comprising the CTP-modified polypeptide and apharmaceutically acceptable carrier.

In one embodiment, the present invention relates to a polynucleotidemolecule comprising a coding portion encoding a CTP-modified polypeptideconsisting of a coagulation factor and three chorionic gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid coagulation factor. In one embodiment, the sequence of at least oneCTP is selected from the group consisting of: SEQ ID NO: 1 and SEQ IDNO: 2. In one embodiment, at least one CTP is truncated. In oneembodiment, the coagulation factor is a vitamin K dependentglycoprotein. In another embodiment, the coagulation factor is FactorIX. In still another embodiment, the coagulation factor is Factor VII.In yet another embodiment the coagulation factor is activated FVII(FVIIa).

In one embodiment, the present invention relates to an expression vectorcomprising a polynucleotide molecule described herein.

In one embodiment, the present invention relates to a cell comprising anexpression vector described herein.

In one embodiment, the present invention relates to a compositioncomprising an expression vector described herein.

In one embodiment, the present invention relates to a method ofextending the biological half-life of a coagulation factor, comprisingthe step of attaching three chorionic gonadotropin carboxy terminalpeptides to the carboxy terminus of the coagulation factor, therebyextending the biological half-life of the coagulation factor. In oneembodiment, at least one CTP is encoded by an amino acid sequenceselected from the group consisting of: SEQ ID NO:1 and SEQ ID NO: 2. Inone embodiment, at least one CTP is glycosylated. In one embodiment, atleast one CTP is truncated. In one embodiment, at least one CTP isattached to the coagulation factor via a linker. In one embodiment, thelinker is a peptide bond.

In one embodiment, the present invention relates to a method ofimproving the area under the curve (AUC) of a coagulation factor,comprising the step of attaching three chorionic gonadotropin carboxyterminal peptides to the carboxy terminus of the coagulation factor,thereby improving the AUC of the coagulation factor. In one embodiment,at least one CTP is encoded by an amino acid sequence selected from thegroup consisting of: SEQ ID NO:1 and SEQ ID NO: 2. In one embodiment, atleast one CTP is glycosylated. In one embodiment, at least one CTP istruncated. In one embodiment, at least one CTP is attached to thecoagulation factor via a linker. In one embodiment, the linker is apeptide bond.

In one embodiment, the present invention relates to a method of reducingthe dosing frequency of a coagulation factor, comprising the step ofattaching three chorionic gonadotropin carboxy terminal peptides to thecarboxy terminus of the coagulation factor, thereby reducing the dosingfrequency of the coagulation factor. In one embodiment, at least one CTPis encoded by an amino acid sequence selected from the group consistingof: SEQ ID NO:1 and SEQ ID NO: 2. In one embodiment, at least one CTP isglycosylated. In one embodiment, at least one CTP is truncated. In oneembodiment, at least one CTP is attached to said coagulation factor viaa linker. In one embodiment, the linker is a peptide bond.

In one embodiment, the present invention relates to a method ofpreventing or treating a blood clotting or coagulation disorder in asubject, the method comprising the step of administering a CTP-modifiedcoagulation factor polypeptide of this invention, as described herein,to the subject, thereby preventing or treating a blood clotting orcoagulation disorder in said subject. In one embodiment, disorder ishemophilia. In one embodiment, the subject is a human child. In oneembodiment, administering is via the subcutaneous route.

Other features and advantages of the present invention will becomeapparent from the following detailed description, examples and figures.It should be understood, however, that the detailed description and thespecific examples while indicating preferred embodiments of theinvention are given by way of illustration only, since various changesand modification within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A. Shows a bar graph showing harvests limited, diluted,transfected, and selected cells with FIX-CTP and FIX-CTP-CTP variants inthe presence of 5 μg/ml of Vitamin K3. The level of FIX was quantifiedusing Human FIX ELISA kit (Affinity Biologicals; Cat. No. FIX-AG RUO),and the calculated protein concentration (μg/ml) is the average of twoindependent runs.

FIG. 1B. Shows SDS-PAGE gel micrographs of FIX Ab recognition anddepicts recognition of anti-FIX antibody in Western-blot; Lane 1 in FIG.1B was loaded with a sample containing recombinant FIX; Lane 2 in FIG.1B was loaded with a sample containing FIX-CTP harvests. Lane 3 in FIG.1B was loaded with a sample containing FIX-(CTP)₂ harvest.

FIG. 1C. Shows SDS-PAGE gel micrographs of FIX Ab recognition. FIG. 1Cdepicts recognition of anti-γ carboxylation antibody in Western-blot.Lane 1 in FIG. 1C was loaded with a sample containing recombinant FIX.Lane 2 in FIG. 1C was loaded with a sample containing FIX-CTP harvests.Lane 3 in FIG. 1C was loaded with a sample containing FIX-(CTP)₂harvest.

FIG. 2. Shows a graph showing FIX-CTP and FIX-(CTP)₂ harvestscomparative chromogenic activity (measured by a the EC₅₀. concentration)compared to rhFIX (American Diagnostics).

FIG. 3. Shows a graph showing PK profile of rhFIX, harvest ofFIX-CTP-CTP, and harvest of FIX-CTP.

FIG. 4. Shows a bar graph showing harvests of FIX-CTP and FIX-CTP-CTPharvests and FIX-CTP-CTP purified protein FIX antigen level asdetermined using Human FIX ELISA kit (Affinity Biologicals; cat. No.FIX-AG RUO). The calculated protein concentration (μg/ml) is the averageof two independent runs.

FIG. 5A. Shows SDS-PAGE gel micrographs of FIX Ab recognition anddepicts a coomassie blue staining. Lane 1 was loaded with a samplecontaining FIX-(CTP)₂. Lane 2 was loaded with a sample containingunbound FIX-(CTP)₂. Lane 3 was loaded with a sample containing aconcentrated elution of FIX-(CTP)₂.

FIG. 5B. Shows SDS-PAGE gel micrographs of FIX Ab recognition anddepicts recognition of anti-FIX antibody in Western-blot. Lane 1 wasloaded with a sample containing FIX-(CTP)₂. Lane 2 was loaded with asample containing unbound FIX-(CTP)₂. Lane 3 was loaded with a samplecontaining a concentrated elution of FIX-(CTP)₂.

FIG. 5C. Shows SDS-PAGE gel micrographs of FIX Ab recognition anddepicts recognition of anti-γ carboxylation antibody in Western-blot.Lane 1 was loaded with a sample containing FIX-(CTP)₂. Lane 2 was loadedwith a sample containing unbound FIX-(CTP)₂. Lane 3 was loaded with asample containing a concentrated elution of FIX-(CTP)₂.

FIG. 6. Shows a graph showing FIX-(CTP)₂ chromogenic activity (sampleconcentration/O.D.) compared to human normal pool plasma and rhFIX(American Diagnostics).

FIG. 7. Shows a graph showing the PK profile of purified FIX-CTP-CTP,rhFIX, harvest of FIX-CTP-CTP, and harvest of FIX-CTP.

FIG. 8A. Shows an anti-CTP and anti-gamma carboxylation antibodiesWestern blots of FIX fused to three, four or five CTPs. FIX-CTP₃,FIX-CTP₄, and FIX-CTP₅ harvests were loaded on 12% Tris-Glycine gelusing Precision plus dual color protein marker (Bio-Rad). The SDS-PAGEanalysis was performed by Western immuno-blot using anti-CTP polyclonalAb (Adar Biotech Production).

FIG. 8B. Shows an anti-CTP and anti-gamma carboxylation antibodiesWestern blots of FIX fused to three, four or five CTPs. FIX-CTP₃,FIX-CTP₄, and FIX-CTP₅ harvests were loaded on 12% Tris-Glycine gelusing Precision plus dual color protein marker (Bio-Rad). The SDS-PAGEanalysis was performed by Western immuno-blot using anti-Gla Ab(American Diagnostica).

FIG. 9. Shows a Coomassie blue detection of FIX-CTP₃, FIX-CTP₄, andFIX-CTP₅. After a purification process utilizing Jacalin column(immunoaffinity purification of glycosylated proteins), FIX-CTP₃,FIX-CTP₄, and FIX-CTP₅ were loaded on 12% Tris-Glycine gel usingPrecision Plus Dual Color Protein Marker (Bio-Rad). The SDS-PAGE wasstained by Coomassie blue dye for sample detection.

FIG. 10. Shows FIX Chromogenic activity. A comparative assessment of thein vitro potency of fully purified (HA column) FIX-CTP₃ FIX-CTP₄ andFIX-CTP₅ versus human pool normal plasma was performed using acommercially available chromogenic activity test kit, BIOPHEN (HyphenBioMed 221802). All samples were serially diluted and the potency wasassessed by comparing a dose response curve to a reference preparationconsisting of normal human plasma.

FIG. 11. Shows the comparative pharmacokinetic (PK) profile of FIX-CTP₃FIX-CTP₄ and FIX-CTP₅. FIX concentration in plasma samples werequantified using human FIX Elisa kits (Affinity Biologicals).Pharmacokinetic profile was calculated and is the mean of 3 animals ateach time point. Terminal half-lives were calculated using PK Solutions2.0 software.

FIG. 12A. Shows the FIX-CTP₃ SDS-PAGE analysis—Coomassie SDS-PAGE.FIX-CTP₃ γ-carboxylated enriched protein, rhFIX and rFIXa (activatedFIX) were loaded on 12% Tris-Glycine gel using Precision Plus Dual ColorProtein Marker (Bio-Rad). The SDS-PAGE Coomassie analysis was performedby staining the gel with Coomassie blue reagent (800 ng of protein).

FIG. 12B. Shows the FIX-CTP₃ SDS-PAGE analysis—Coomassie SDS-PAGE.FIX-CTP₃ γ-carboxylated enriched protein, rhFIX and rFIXa (activatedFIX) were loaded on 12% Tris-Glycine gel using Precision Plus Dual ColorProtein Marker (Bio-Rad). A Western immunoblot was performed using 100ng of protein with anti-human FIX polyclonal Ab.

FIG. 12C. Shows the FIX-CTP₃ SDS-PAGE analysis—Coomassie SDS-PAGE.FIX-CTP₃ γ-carboxylated enriched protein, rhFIX and rFIXa (activatedFIX) were loaded on 12% Tris-Glycine gel using Precision Plus Dual ColorProtein Marker (Bio-Rad). A Western immunoblot was performed using 100ng of protein with anti-human gamma carboxylation monoclonal antibody(American Diagnostics Cat #499, 3570).

FIG. 12D. Shows the FIX-CTP₃ SDS-PAGE analysis—Coomassie SDS-PAGE.FIX-CTP₃ γ-carboxylated enriched protein, rhFIX and rFIXa (activatedFIX) were loaded on 12% Tris-Glycine gel using Precision Plus Dual ColorProtein Marker (Bio-Rad). A Western immunoblot was performed using 100ng of protein with anti-FIX pro-peptide polyclonal Ab (FIG. 12D).

FIG. 12E. Shows the FIX-CTP₃ SDS-PAGE analysis—Coomassie SDS-PAGE.FIX-CTP₃ γ-carboxylated enriched protein, rhFIX and rFIXa (activatedFIX) were loaded on 12% Tris-Glycine gel using Precision Plus Dual ColorProtein Marker (Bio-Rad). A Western immunoblot was performed using 100ng of protein with anti-CTP polyclonal Ab.

FIG. 13. Shows the FIX-CTP₃ chromogenic activity. A comparativeassessment of the in vitro potency of FIX-CTP₃ harvest and FIX-CTP₃γ-carboxylated enriched protein, versus human pool normal plasma wasperformed using a commercially available chromogenic activity test kit,BIOPHEN (Hyphen BioMed 221802). FIX-CTP₃ harvest and protein wereserially diluted, and the potency was assessed by comparing adose-response curve to a reference preparation consisting of normalhuman plasma.

FIG. 14. Shows the comparative clotting time. An in vitro aPTT(activated Partial Thrombin Time Assay) was performed comparing theclotting activity of FIX-CTP₃ to BeneFIX. The proteins were seriallydiluted and spiked into human FIX-depleted plasma, and the clotting timewas evaluated.

FIG. 15. Shows FIX-CTP₃ comparative PK profile. FIX concentration wasquantitated using human FIX ELISA kits (Affinity Biologicals; Cat. #FIX-AG RUO). The pharmacokinetic profile was calculated for each proteinand is the mean of 3 animals at each time point.

FIG. 16A. In parallel to PK sampling, FIX-deficient animals administeredwith FIX-CTP₃, citrated plasma samples, were evaluated for theirclotting activity by aPTT assay, which was translated to % activity. The% activity at each collection point was calculated as the currentclotting time/clotting time of normal pool mice plasma* 100.

FIG. 16B. In parallel to PK sampling, FIX-deficient animals administeredwith either BeneFIX®, citrated plasma samples, were evaluated for theirclotting activity by aPTT assay, which was translated to % activity. The% activity at each collection point was calculated as the currentclotting time/clotting time of normal pool mice plasma* 100.

FIG. 17A. Shows a first challenge bleeding parameters. FIX-deficientmice were administered a single intravenous injection of 100 IU/Kg ofBeneFIX® or rFIX-CTP₃. The tail vein was slightly clipped 48 hourspost-dosing and tail vein bleeding time (TVBT) was evaluated. A secondbleeding challenge was performed 15 minutes after reaching homeostasis,and the same parameters were measured.

FIG. 17B. Shows a first challenge bleeding parameters. FIX-deficientmice were administered a single intravenous injection of 100 IU/Kg ofBeneFIX® or rFIX-CTP₃. The tail vein was slightly clipped 48 hourspost-dosing and tail vein bleeding time (TVBT) was evaluated. A secondbleeding challenge was performed 15 minutes after reaching homeostasis,and the same parameters were measured.

FIG. 17C. Shows a first challenge bleeding parameters. FIX-deficientmice were administered a single intravenous injection of 100 IU/Kg ofBeneFIX® or rFIX-CTP₃. The tail vein was slightly clipped 48 hourspost-dosing and bleeding intensity (hemoglobin OD) was evaluated. Asecond bleeding challenge was performed 15 minutes after reachinghomeostasis, and the same parameters were measured.

FIG. 17D. Shows a first challenge bleeding parameters. FIX-deficientmice were administered a single intravenous injection of 100 IU/Kg ofBeneFIX® or rFIX-CTP₃. The tail vein was slightly clipped 48 hourspost-dosing and bleeding intensity (hemoglobin OD) was evaluated. Asecond bleeding challenge was performed 15 minutes after reachinghomeostasis, and the same parameters were measured.

FIG. 18A. Shows a second challenge bleeding parameters. Once the firstbleeding described in the legend to FIG. 19 was spontaneously ormanually stopped, a second bleeding challenge was performed 15 minutesfollowing the first one, and the time was re-measured.

FIG. 18B. Shows a second challenge bleeding parameters. Once the firstbleeding described in the legend to FIG. 19 was spontaneously ormanually stopped, a second bleeding challenge was performed 15 minutesfollowing the first one, and the time was re-measured.

FIG. 18C. Shows a second challenge bleeding parameters. Once the firstbleeding described in the legend to FIG. 19 was spontaneously ormanually stopped, a second bleeding challenge was performed 15 minutesfollowing the first one, and the bleeding intensity was re-measured.

FIG. 18D. Shows a second challenge bleeding parameters. Once the firstbleeding described in the legend to FIG. 19 was spontaneously ormanually stopped, a second bleeding challenge was performed 15 minutesfollowing the first one, and the bleeding intensity was re-measured.

FIG. 19A. Shows a diagram illustrating the rFVII-CTP construct.

FIG. 19B. Shows a diagram illustrating the rFVII-CTP-CTP construct.

FIG. 19C. Shows a diagram illustrating the rFIX-CTP construct.

FIG. 19D. Shows a diagram illustrating the rFIX-CTP-CTP construct.

FIG. 20A. Shows a bar graph showing harvests limited diluted clonetransfected and selected cells with FVII-CTP variants in the presence of5 μg/ml of Vitamin K3. The level of FVII was quantified using FVII ELISA(AssayPro).

FIG. 20B. Shows a bar graph showing harvests of limited dilutedtransfected and selected cells with FVII-CTP variants in the presence of5 μg of Vitamin K3.activity. FVII activity was quantified using FVIIchromogenic activity assay (AssayPro).

FIG. 20C. Shows a bar graph showing harvests of limited dilutedtransfected and selected cells with FVII-CTP variants in the presence of5 μg of Vitamin K3. The specific activity of FVII was calculated foreach version by dividing the activity value by the harvest FVIIconcentration.

FIG. 20D. Shows a graph showing PK profile of FVII, FVII-CTP-CTP, andFVII-CTP harvests.

FIG. 21A. Shows western blots of FVII fused to three, four and fiveCTPs, detected using anti-FVII, anti-CTP, and anti-gamma carboxylationantibodies. FVII-CTP₃, FVII-CTP₄, and FVII-CTP₅ harvests were loaded on12% Tris-Glycine gel (expedeon) using Precision plus dual color proteinmarker (Bio-Rad). The SDS-PAGE analysis was performed by Westernimmunoblot using anti-FVII.

FIG. 21B. Shows western blots of FVII fused to three, four and fiveCTPs, detected using anti-FVII, anti-CTP, and anti-gamma carboxylationantibodies. FVII-CTP₃, FVII-CTP₄, and FVII-CTP₅ harvests were loaded on12% Tris-Glycine gel (expedeon) using Precision plus dual color proteinmarker (Bio-Rad). The SDS-PAGE analysis was performed by Westernimmunoblot using anti-CTP polyclonal Ab (Adar Biotech Production).

FIG. 21C. Shows western blots of FVII fused to three, four and fiveCTPs, detected using anti-FVII, anti-CTP, and anti-gamma carboxylationantibodies. FVII-CTP₃, FVII-CTP₄, and FVII-CTP₅ harvests were loaded on12% Tris-Glycine gel (expedeon) using Precision plus dual color proteinmarker (Bio-Rad). The SDS-PAGE analysis was performed by Westernimmunoblot using anti-Gla Ab (American Diagnostica).

FIG. 22. Shows the FVII Activity—Chromogenic activity. A comparativeassessment of the in vitro potency of HA purified (highly gammacarboxylated fraction) FVII-CTP₃, FVII-CTP₄, and FVII-CTP₅ versus normalhuman pool plasma was performed using a commercially availablechromogenic activity test kit, BIOPHEN (Hyphen BioMed 221304). Allsamples were serially diluted and the potency was assessed by comparinga dose response curve to a reference preparation consisting of normalhuman plasma.

FIG. 23. Shows a first comparative pharmacokinetic (PK) profile-FVII 3,4 and 5 CTPs. FVII-CTP₃, FVII-CTP₄, and FVII-CTP₅ (Group A, B and C,respectively) were administered in a single intravenous injection toSprague Dawley rats (six rats per treatment) in a dose of 250 μg/kg bodyweight. Blood samples were drawn retro-orbitally from 3 rats alternatelyat 0.083, 0.5 2, 5, 8, 24, 48, 72 and 96 hours post dosing. Citratedplasma (0.38%) was prepared immediately after sampling and stored at−20° C. until analysis. FVII-CTP₅ demonstrated a superior profile ascompared to the two other versions.

FIG. 24. Shows a second comparative PK profile-FVII 3, 4 and 5 CTPs.FVII-CTP₃, FVII-CTP₄, and FVII-CTP₅ following FVII selection and the HApurification process (Group A, B and C, respectively) were administeredin a single intravenous injection to Sprague Dawley rats (three rats persubstance) in a dose of 29.45 μg/kg body weight. Blood samples weredrawn retro-orbital at 0.083, 0.5 2, 8, 24, 48, and 72 hourspost-dosing. Citrated plasma (0.38%) was prepared immediately aftersampling and stored at −20° C. until analysis.

FIG. 25A. Shows a schematic diagram of FVII-CTP₃ purification process.Batch 31 was produced for the PK/PD study.

FIG. 25B. Shows a schematic diagram of FVII-CTP₃ purification process.Batch 38 was produced for the survival study.

FIG. 26A. Shows an SDS-PAGE and Western blot of Final FVII and FVIIa. 10μg (Batch 31) or 5 μg (Batch 38) were loaded in each lane of Coomassiestained SDS-PAGE. 1. FVII-CTP₃ polypeptide; 2. Heavy chain, including3×CTP; 3. Light Chain. All three antibodies detect FVII.

FIG. 26B. Shows an SDS-PAGE and Western blot of Final FVII and FVIIa. 10μg (Batch 31) or 5 μg (Batch 38) were loaded in each lane of Coomassiestained SDS-PAGE 1. FVII-CTP₃ polypeptide; 2. Heavy chain, including3×CTP; 3. Light Chain.

FIG. 26C. Shows an SDS-PAGE and Western blot of Final FVII and FVIIa. 10μg (Batch 31) or 5 μg (Batch 38) were loaded in each lane of Coomassiestained SDS-PAGE 1. FVII-CTP₃ polypeptide; 2. Heavy chain, including3×CTP; 3. Light Chain.

FIG. 26D. Shows an SDS-PAGE and Western blot of Final FVII and FVIIa. 10μg (Batch 31) or 5 μg (Batch 38) were loaded in each lane of Coomassiestained SDS-PAGE 1. FVII-CTP₃ polypeptide; 2. Heavy chain, including3×CTP; 3. Light Chain.

FIG. 26E. Shows an SDS-PAGE and Western blot of Final FVII and FVIIa. 10μg (Batch 31) or 5 μg (Batch 38) were loaded in each lane of Coomassiestained SDS-PAGE 1. FVII-CTP₃ polypeptide; 2. Heavy chain, including3×CTP; 3. Light Chain.

FIG. 26F. Shows an SDS-PAGE and Western blot of Final FVII and FVIIa. 1μg protein was loaded in each lane of Western blot. 1. FVII-CTP₃polypeptide; 2. Heavy chain, including 3×CTP; 3. Light Chain. All threeantibodies detect FVII. FVIIa light chain is detected with both α-FVII.

FIG. 26G. Shows an SDS-PAGE and Western blot of Final FVII and FVIIa. 1μg protein was loaded in each lane of Western blot. 1. FVII-CTP₃polypeptide; 2. Heavy chain, including 3×CTP; 3. Light Chain. All threeantibodies detect FVII. FVIIa heavy chain was detected by α-CTP.

FIG. 26H. Shows an SDS-PAGE and Western blot of Final FVII and FVIIa. 1μg protein was loaded in each lane of Western blot. 1. FVII-CTP₃polypeptide; 2. Heavy chain, including 3×CTP; 3. Light Chain. All threeantibodies detect FVII. FVIIa heavy chain was detected by α-Gla.

FIG. 27. Shows that FVII-CTP₃ chromogenic activity is enhanced as aresult of purification on ceramic hydroxyapatite (HA) column. Acomparative assessment of the in vitro potency of FVII-CTP₃ harvest,in-process fractions, and purified FVII-CTP₃ versus human pool normalplasma was performed using a commercially available chromogenic activitytest kit, BIOPHEN (Hyphen BioMed 221304). FVII-CTP₃ harvest and proteinwere serially diluted and the potency was assessed by comparing adose-response curve to a reference preparation of normal human plasma.

FIG. 28. Shows the PK profile of FVIIa-CTP₃ vs. NovoSeven® inFVIII-deficient mice. FVIIa-CTP₃ was produced following FVII selection,HA purification process and activation. FVIIa-CTP₃ or NovoSeven® wasadministered in a single intravenous injection to FVIII−/− hemophilicmice. Blood samples were drawn retro-orbitally at 0.083, 0.5 2, 8, 24,48, and 72 hours post-dosing. Citrated plasma (0.38%) was preparedimmediately after sampling and stored at −20° C. until analysis, and aPK profile was established based on FVIIa clotting activity using aSTACLOT commercial kit.

FIG. 29A. Shows that FVIIa-CTP₃ was produced following FVII selection,HA purification process and activation. FVIIa-CTP₃ or NovoSeven® wasadministered in a single intravenous injection to FVIII−/− hemophilicmice. Blood samples were drawn retro-orbitally at 0.083, 0.5 2, 8, 24,48, and 72 hours post-dosing. Citrated plasma (0.38%) was preparedimmediately after sampling and stored at −20° C. until analysis.Thrombin generation parameters were evaluated during the PK experiment,and parameters including maximal amount to peak was evaluated.

FIG. 29B. Shows that FVIIa-CTP₃ was produced following FVII selection,HA purification process and activation. FVIIa-CTP₃ or NovoSeven® wasadministered in a single intravenous injection to FVIII−/− hemophilicmice. Blood samples were drawn retro-orbitally at 0.083, 0.5 2, 8, 24,48, and 72 hours post-dosing. Citrated plasma (0.38%) was preparedimmediately after sampling and stored at −20° C. until analysis.Thrombin generation parameters were evaluated during the PK experiment,and parameters including amount of thrombin to time point was evaluated.

FIG. 29C. Shows that FVIIa-CTP₃ was produced following FVII selection,HA purification process and activation. FVIIa-CTP₃ or NovoSeven® wasadministered in a single intravenous injection to FVIII−/− hemophilicmice. Blood samples were drawn retro-orbitally at 0.083, 0.5 2, 8, 24,48, and 72 hours post-dosing. Citrated plasma (0.38%) was preparedimmediately after sampling and stored at −20° C. until analysis.Thrombin generation parameters were evaluated during the PK experiment,and parameters including rate of thrombin generation was evaluated.

FIG. 30A. Shows hemophilic mice survival curves post tail vaintransection (TVT). TVT was performed 15 min post administration. MiceSurvival was observed for 24 hours after TVT and recorded every singlehour for the first 12 hours, and after 24 hours. Control group data(vehicle) is the sum of the 3 experiments with 5 mice/experiment.

FIG. 30B. Shows hemophilic mice survival curves post tail vaintransection (TVT). TVT was performed 24 hours post administration. MiceSurvival was observed for 24 hours after TVT and recorded every singlehour for the first 12 hours, and after 24 hours. Control group data(vehicle) is the sum of the 3 experiments with 5 mice/experiment.

FIG. 30C. Shows hemophilic mice survival curves post tail vaintransection (TVT). TVT was performed 48 hours post administration. MiceSurvival was observed for 24 hours after TVT and recorded every singlehour for the first 12 hours, and after 24 hours. Control group data(vehicle) is the sum of the 3 experiments with 5 mice/experiment.

FIG. 30D. Summarizes mouse survival as recorded 24 hours post TVT.

FIG. 31A. Shows FVII-3-CTP and FVII-5 CTP immune-blots, blotted for GLA.

FIG. 31B. Shows FVII-3-CTP and FVII-5 CTP immune-blots, blotted forFVII.

FIG. 31C. Shows FVII-3-CTP and FVII-5 CTP immune-blots, blotted for CTP.

FIG. 32. Shows a comparative PK profile-FVII 3 & 5 CTP- from select andHA column purification (FVIIS vs. FVII HA).

FIG. 33. Shows a comparative PK profile-FVII 3 & 5 CTP—The second study(IV vs. SC).

FIG. 34. Shows hemophilic mice survival curves post tail vaintransection (TVT) following SC administration. TVT was performed 12hours post administration. Mice Survival was observed for 24 hours afterTVT and recorded every single hour for the first 12 hours, and after 24hours.

FIG. 35A. Shows the PK profile of MOD-5014 vs. NovoSeven® following IVadministration.

FIG. 35B. Shows the PK profile of MOD-5014 vs. NovoSeven® following SCadministration.

FIG. 36. Shows the PK profile of MOD-5014 (Clone 61 #75, #81) vs.NovoSeven® following single SC administration.

FIG. 37. Shows that warfarin increases PT and aPTT values. SD-ratsreceived 10 mg/Kg warfarin per-os, and blood samples were taken at thedesignated time point. Plasma was prepared and PT and aPTT values weredetermined.

FIG. 38. Acute effect of IV injection of MOD-5014 and NovoSeven® onWarfarin treated rats.

FIG. 39. Shows the response of Warfarin treated rats to a wide range ofMOD-5014 and NovoSeven® doses, 24 hours post injection.

FIG. 40. Shows that MOD-5014 restored PT values to normal up to 48 hourspost dosing, while the effect of NovoSeven® no longer exists after 24hours.

FIG. 41. Shows IV injection of MOD-5014 reduce bleeding time in warfarintreated rats as compared to NovoSeven® 24 and 48 hours post injection.

FIG. 42. Shows that MOD-5014 is able to restore PT values to normal upto 48 hours post dosing, while the effect of NovoSeven® no longer existsafter 24 hours

FIG. 43. Shows superiority over NovoSeven® by keeping the blood loss atlow level for 48 hours after administration.

FIG. 44. Shows that Tissue Factor Pathway Inhibitor (TFPI) inhibitsMOD-5014 and NovoSeven® in a similar dose-dependent manner.

FIG. 45. Shows that anti-thrombin III inhibited MOD-5014 and NovoSeven®in a similar manner.

FIG. 46. Shows the results of Factor X activation by MOD-5014 andNovoSeven®, which were almost identical.

FIG. 47. Shows the Factor X activation in the presence of TFPI (20 μg/mlto 0.002 ng/ml) by MOD-5014 and NovoSeven®, present at 0.6 ng/ml.

FIG. 48. Shows the Factor X activation in the presence of TFPI (20 μg/mlto 0.002 ng/ml) by MOD-5014 and NovoSeven®, present at 4.0 ng/ml.

FIG. 49. Shows Factor X activation in the presence of TFPI and Heparin,wherein MOD-5014 and NovoSeven® exhibited similar activation.

FIG. 50. Shows Factor X activation in the presence of anti-thrombin III,wherein MOD-5014 and NovoSeven® exhibited similar activation.

FIG. 51. Shows the Factor X activation by MOD-5014 and NovoSeven® in thepresence of Heparin, wherein similar moderate inhibition was observed.

FIG. 52. Shows similar Factor X activation by MOD-5014 and NovoSeven® inthe presence of anti-thrombin and heparin.

FIG. 53. Shows a MOD-5014 thrombin generation profile as compared tocommercially available NovoSeven® at high phospholipid (PL)concentration.

FIG. 54. Shows a MOD-5014 peak thrombin generation profile as comparedto commercially available NovoSeven® at low phospholipid (PL)concentration.

FIGS. 55A and B. Show the thromboelastography results for MOD-5014 andNovoSeven®, wherein both decreased clotting time and increased the rateof clot formation.

FIG. 56. Shows the results of NovoSeven® thrombin generation (TG)following re-calcification. (Run #1)

FIG. 57. Shows the results of MOD-5014 thrombin generation (TG)following re-calcification. (Run #1)

FIG. 58 A-E. Provide an overlay analysis of NovoSeven® (NS) resultsversus MOD-5014 (PRO) TG results at similar concentrations. (A) providesresults at 1.25 μg/ml. (B) provides results at 5 μg/ml. (C) providesresults at 15 μg/ml. (D) provides results at 2.5 μg/ml. (E) providesresults at 10 μg/ml. (Run #1)

FIG. 59 A-D. Provide an overlay analysis of NovoSeven® (NS) resultsversus MOD-5014 (PRO) TG results at different concentrations. (A) showsresults of NS at 1.25 μg/ml and PRO at 2.5 μg/ml. (B) shows results ofNS at 5 μg/ml and PRO at 10 μg/ml (C) shows results of NS at 2.5 μg/mland PRO at 5 μg/ml. (D) shows results of NS at 10 μg/ml and PRO at 15μg/ml. (Run #1)

FIG. 60. Shows the results of NovoSeven® (NS) thrombin generation (TG)following re-calcification for NS at different concentrations. (Run #2)

FIG. 61. Shows the results of MOD-5014 (PRO) thrombin generation (TG)following re-calcification for PRO at different concentrations. (Run #2)

FIG. 62 A-E. Provide an overlay analysis of NovoSeven® (NS) resultsversus MOD-5014 (PRO) TG results at similar concentrations. (A) providesresults at 1.25 μg/ml. (B) provides results at 5 μg/ml. (C) providesresults at 15 μg/ml. (D) provides results at 2.5 μg/ml. (E) providesresults at 10 μg/ml. (Run #2)

FIG. 63 A-D. Provide an overlay analysis of NovoSeven® (NS) resultsversus MOD-5014 (PRO) TG results at different concentrations. (A) showsresults of NS at 1.25 μg/ml and PRO at 2.5 μg/ml. (B) shows results ofNS at 5 μg/ml and PRO at 10 μg/ml. (C) shows results of NS at 2.5 μg/mland PRO at 5 μg/ml. (D) shows results of NS at 10 μg/ml and PRO at 15μg/ml. (Run #2)

FIG. 64. Shows data for TG by NovoSeven® following re-calcification atlow TF concentration. (Run #1)

FIG. 65. Shows data for TG by MOD-5014 following re-calcification at lowTF concentration. (Run #1)

FIG. 66 A-E. Provide an overlay analysis of NovoSeven® (NS) resultsversus MOD-5014 (PRO) results at similar concentrations, followingre-calcification at low TF. (A) provides results at 1.25 μg/ml. (B)provides results at 5 μg/ml. (C) provides results at 15 μg/ml. (D)provides results at 2.5 μg/ml. (E) provides results at 10 μg/ml. (Run#1)

FIG. 67. Shows data for TG by NovoSeven® following re-calcification atlow TF concentration. (Run #2)

FIG. 68. Shows data for TG by MOD-5014 following re-calcification at lowTF concentration. (Run #2)

FIG. 69 A-E. Provide an overlay analysis of NovoSeven® (NS) resultsversus MOD-5014 (PRO) results at similar concentrations, followingre-calcification at low TF. (A) provides results at 1.25 μg/ml. (B)provides results at 5 μg/ml. (C) provides results at 15 μg/ml. (D)provides results at 2.5 μg/ml. (E) provides results at 10 μg/ml. (Run#2)

FIG. 70 A-C. Provide an overlay analysis of NovoSeven® (NS) resultsversus MOD-5014 (PRO) results at different concentrations, followingre-calcification at low TF. (A) shows results of NS at 1.25 μg/ml andPRO at 5 μg/ml. (B) shows results of NS at 5 μg/ml and PRO at 15 μg/ml.(C) shows results of NS at 2.5 μg/ml and PRO at 10 μg/ml. (Run #2)

FIG. 71. Shows complete thrombin generation by FVIII, as a comparisonfor the results with NovoSeven® and MOD-5014 in the presence and absenceof low TF.

FIG. 72. Provides an overlay analysis of thrombin generation by FVIII inthe presence and absence of low TF.

FIG. 73. Provides an overlay analysis of NovoSeven® (NS; 1.25 μg/kg) TGresults versus MOD-5014 (PRO; 1.25 μg/kg) TG results at escalatingconcentrations of TF. (Run #1)

FIG. 74 A-E. Provide an overlay analysis of NovoSeven® (NS; 1.25 μg/kg)TG results versus MOD-5014 (PRO; 1.25 μg/kg) TG results at escalatingconcentrations of TF. (A) provides results at 0 pM TF. (B) providesresults at 1 pM TF. (C) provides results at 5 pM TF. (D) providesresults at 0.5 pM TF. (E) provides results at 2.5 pM TF. (Run #1)

FIG. 75. Provides an overlay analysis of NovoSeven® (NS; 2.5 μg/kg) TGresults versus MOD-5014 (PRO; 2.5 μg/kg) TG results at escalatingconcentrations of TF. (Run #1)

FIG. 76 A-E. Provide an overlay analysis of NovoSeven® (NS; 2.5 μg/kg)TG results versus MOD-5014 (PRO; 2.5 μg/kg) results at escalatingconcentrations of TF. (A) provides results at 0 pM TF. (B) providesresults at 1 pM TF. (C) provides results at 5 pM TF. (D) providesresults 0.5 pM TF. (E) provides results at 2.5 pM TF. (Run #1)

FIG. 77. Provides an overlay analysis of NovoSeven® (NS; 5 μg/kg) TGresults versus MOD-5014 (PRO; 5 μg/kg) results at escalatingconcentrations of TF. (Run #1)

FIG. 78 A-E. Provide an overlay analysis of NovoSeven® (NS; 5 μg/kg) TGresults versus MOD-5014 (PRO; 5 μg/kg) TG results at escalatingconcentrations of TF. (A) provides results at 0 pM TF. (B) providesresults at 1 pM TF. (C) provides results at 5 pM TF. (D) providesresults 0.5 pM TF. (E) provides results at 2.5 pM TF. (Run #1)

FIG. 79 A-C. Show a dose dependent TG response in the presence ofNovoSeven® (NS) at escalating concentrations of TF. (A) provides resultsat 1.25 μg/kg NS. (B) provides results at 5 μg/kg NS. (C) providesresults at 2.5 μg/kg NS. (Run #1)

FIG. 80 A-C. Show a dose dependent TG response in the presence ofMOD-5014 (PRO) at escalating concentrations of TF. (A) provides resultsat 1.25 μg/kg PRO. (B) provides results at 5 μg/kg PRO. (C) providesresults at 2.5 μg/kg PRO. (Run #1)

FIG. 81. Provides an overlay analysis of NovoSeven® (NS; 10 μg/kg) TGresults versus MOD-5014 (PRO; 10 μg/kg) TG results at escalatingconcentrations of TF. (Run #2)

FIG. 82. Provides an overlay analysis of NovoSeven® (NS; 2.5 μg/kg) TGresults versus MOD-5014 (PRO; 2.5 μg/kg) TG results at escalatingconcentrations of TF. (Run #2)

FIG. 83. Provides an overlay analysis of NovoSeven® (NS; 5 μg/kg) TGresults versus MOD-5014 (PRO; 5 μg/kg) results at escalatingconcentrations of TF. (Run #2)

FIG. 84 A-C. Show a dose dependent TG response in the presence ofNovoSeven® (NS) at escalating concentrations of TF. (A) provides resultsat 2.5 μg/ml NS. (B) provides results at 10 μg/ml NS. (C) providesresults at 5 μg/ml NS. (Run #2)

FIG. 85 A-C. Show a dose dependent TG response in the presence ofMOD-5014 (PRO) at escalating concentrations of TF. (A) provides resultsat 2.5 μg/ml PRO. (B) provides results at 10 μg/ml PRO. (C) providesresults at 5 μg/ml PRO. (Run #2)

FIG. 86 A-E. Provide an overlay analysis of NovoSeven® (NS; 10 μg/kg) TGresults versus MOD-5014 (PRO; 10 μg/kg) TG results at escalatingconcentrations of TF. (A) provides results at 0 pM TF. (B) providesresults at 1 pM TF. (C) provides results at 5 pM TF. (D) providesresults 0.5 pM TF. (E) provides results at 2.5 pM TF. (Run #2)

FIG. 87. Provides an overlay analysis of NovoSeven® (NS; 2.5 μg/kg) TGresults versus MOD-5014 (PRO; 2.5 μg/kg) TG results at escalatingconcentrations of TF (A) provides results at 0 pM TF. (B) providesresults at 1 pM TF. (C) provides results at 5 pM TF. (D) providesresults 0.5 pM TF. (E) provides results at 2.5 pM TF. (Run #2)

FIG. 88. Provides an overlay analysis of NovoSeven® (NS; 5 μg/kg) TGresults versus MOD-5014 (PRO; 5 μg/kg) TG results at escalatingconcentrations of TF. (A) provides results at 0 pM TF. (B) providesresults at 1 pM TF. (C) provides results at 5 pM TF. (D) providesresults 0.5 pM TF. (E) provides results at 2.5 pM TF. (Run #2)

FIG. 89. Shows graphs of WBCT Following MOD-5014 Spiking Canine Blood.

FIG. 90. Shows a schematic Drawing of Two-compartment PharmacokineticModel.

FIG. 91. Provides a graph of mean Plasma MOD-5014 Concentrations vs TimeFollowing IV Infusion in Dogs.

FIG. 92. Provides a graph of mean Plasma MOD-5014 Activity vs TimeFollowing IV Infusion in Dogs.

FIGS. 93A-B. Show comparison data of MOD-5014 Plasma Concentration andActivity Following IV Infusion of 50 μg/kg in Dogs. FIG. 93(A) shows theresults for dog P14 and FIG. 93(B) shows the results for dog N06.

FIGS. 94A-D. Show comparison data of MOD-5014 Plasma Concentration andActivity Following IV Infusion of 200 μg/kg in Dogs. FIG. 94(A) showsthe results for dog Blondie;

FIG. 94(B) for dog Josie; FIG. 94(C) for dog N06; and FIG. 94(D) for dogP14.

FIGS. 95A-B. Show comparison data of MOD-5014 Plasma Concentration andActivity Following IV Infusion of 400 μg/kg in Dogs. FIG. 95(A) showsthe results for dog Blondie and FIG. 95(B) shows the results for dogJosie.

FIGS. 96A-B. Show comparison data of MOD-5014 Plasma Concentration andActivity Following IV Infusion of 600 μg/kg in Dogs. FIG. 96(A) showsthe results for dog N05 and

FIG. 96(B) shows the results for dog Joanie.

FIGS. 97A-J. Show plots of Plasma MOD-5014 Concentrations versus Time.Points represent observed plasma concentrations and the line representsthe terminal slope used to calculate T_(1/2). FIG. 97(A) shows theresults for dog N06 after more than 30 hours; FIG. 97(B) for dog P14after more than 30 hours; FIG. 97(C) for dog Blondie after about 50hours; FIG. 97(D) for dog Josie after about 50 hours; FIG. 97(E) for dogN06 after almost 100 hours; FIG. 97(F) for dog P14 after almost 100hours; FIG. 97(G) for dog Blondie after almost 100 hours; FIG. 97(H) fordog Josie after almost 100 hours; FIG. 97(I) for dog Joanie after almost100 hours and FIG. 97(J) for dog N05 after almost 100 hours.

FIGS. 98A-J. Show plots of plasma MOD-5014 Activity versus Time. Pointsrepresent observed plasma concentrations and the line represents theterminal slope used to calculate T₁₁₂. FIG. 98(A) shows the results fordog N06 after more than 30 hours; FIG. 98(B) for dog P14 after more than30 hours; FIG. 98(C) for dog Blondie after almost 50 hours; FIG. 98(D)for dog Josie after almost 50 hours; FIG. 98(E) for dog N06 after almost50 hours; FIG. 98(F) for dog P14 after almost 50 hours; FIG. 98(G) fordog Blondie after almost 50 hours; FIG. 98(H) for dog Josie after almost50 hours; FIG. 98(I) for dog Joanie after almost 50 hours and FIG. 98(J)for dog N05 after almost 50 hours.

FIGS. 99A-J. Show the results of modeling—Plasma Concentrations. Pointsrepresent observed plasma concentrations and the solid line representsconcentrations predicted by model. FIG. 99(A) shows the observed andpredicted results for dog N06; FIG. 99(B) for dog P14; FIG. 99(C) fordog Blondie; FIG. 99(D) for dog Josie; FIG. 99(E) for dog N06 afteralmost 100 hours; FIG. 99(F) for dog P14 after almost 100 hours; FIG.99(G) for dog Blondie after almost 100 hours; FIG. 99(H) for dog Josieafter almost 100 hours; FIG. 99(I) for dog Joanie after almost 100 hoursand FIG. 99(J) for dog N05 after almost 100 hours.

FIGS. 100A-J. Show the results of modeling—Activity. Points representobserved plasma activity and the solid line represents activitypredicted by model. FIG. 100(A) shows the observed and predicted resultsfor dog N06 after at least 32 hours; FIG. 100(B) for dog P14 after atleast 32 hours; FIG. 100(C) for dog Blondie after at least 48 hours;FIG. 100(D) for dog Josie after at least 48 hours; FIG. 100(E) for dogN06 after at least 48 hours; FIG. 100(F) for dog P14 after at least 48hours; FIG. 100(G) for dog Blondie after at least 48 hours; FIG. 100(H)for dog Josie after at least 48 hours; FIG. 100(I) for dog Joanie afterat least 48 hours and FIG. 100(J) for dog N05 after at least 48 hours.

FIG. 101A-D. Show the dose-dependent change in kaolin-initiated TEGkinetics following administration of 50, 200 or 400 μg/kg MOD-5014 indog N06. (A) shows the R-time (Reaction Time); (B) shows the K-time(time from the end of R until the clot reaches 20 mm, the speed of clotformation); (C) shows the Angle (the tangent of the curve made as the Kis reached); and (D) shows the MA (Maximum Amplitude).

FIG. 102A-D. Show the dose-dependent change in kaolin-initiated TEGkinetics following administration of 50 and 200 μg/kg MOD-5014 in dogP-14. (A) shows the R-time (Reaction Time; (B) shows the K-time (timefrom the end of R until the clot reaches 20 mm, the speed of clotformation); (C) shows the Angle (the tangent of the curve made as the Kis reached); and (D) shows the MA (Maximum Amplitude).

FIG. 103A-D. Show the dose-dependent change in kaolin-initiated TEGkinetics following administration of 200 and 400 μg/kg MOD-5014 in dogBlondie. (A) shows the R-time (Reaction Time); (B) shows the K-time(time from the end of R until the clot reaches 20 mm, the speed of clotformation); (C) shows the Angle (the tangent of the curve made as the Kis reached); and (D) shows the MA (Maximum Amplitude).

FIG. 104A-D. Show the dose-dependent change in kaolin-initiated TEGkinetics following administration of 200 and 400 μg/kg MOD-5014 in dogJosie. (A) shows the R-time (Reaction Time); (B) shows the K-time (timefrom the end of R until the clot reaches 20 mm, the speed of clotformation); (C) shows the Angle (the tangent of the curve made as the Kis reached); and (D) shows the MA (Maximum Amplitude).

FIG. 105A-D. Show the dose-dependent change in kaolin-initiated TEGkinetics following administration of 50, 200 or 400 μg/kg MOD-5014 indog Joanie. (A) shows the R-time (Reaction Time); (B) shows the K-time(time from the end of R until the clot reaches 20 mm, the speed of clotformation); (C) shows the Angle (the tangent of the curve made as the Kis reached); and (D) shows the MA (Maximum Amplitude).

FIG. 106A-D. Show the kinetics of TEG over time following MOD-5014administration in dog NOS. (A) shows the R-time (Reaction Time); (B)shows the K-time (time from the end of RT until the clot reaches 20 mm,the speed of clot formation); (C) shows the Angle (the tangent of thecurve made as the K is reached); and (D) shows the MA (MaximumAmplitude).

FIG. 107 A-D. Show the change in TEG performance followingadministration of 270 μg/kg of rhFVIIa (A) shows R-Time (Reaction Time).(B) shows Angle (the tangent of the curve made as the K is reached). (C)shows K-Time (time from the end of RT until the clot reaches 20 mm, thespeed of clot formation). (D) shows the MA (Maximum Amplitude).

FIG. 108A-D. Show MOD-5014 extended TEG effect, representative data froman individual animal (Blondie). (A) shows R-Time (Reaction Time). (B)shows K-Time (time from the end of RT until the clot reaches 20 mm, thespeed of clot formation). (C) shows the Angle (the tangent of the curvemade as the K is reached). (D) shows the MA (Maximum Amplitude). Arrowsin (B), (C) and (D) are pointing to MOD-5014 TEG values 4 hours postdosing.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In one embodiment, the present invention provides long-actingcoagulation factors and methods of producing and using same. In anotherembodiment, long-acting coagulation factors comprise a carboxy terminalpeptide (CTP, also referred to as CTP unit). In another embodiment,long-acting polypeptides which comprise a coagulation factor furthercomprise a carboxy terminal peptide (CTP) of human ChorionicGonadotropin (hCG). In another embodiment, CTP acts as a protectantagainst the degradation of a coagulation factor. In another embodiment,CTP extends the C_(max) of a coagulation factor. In another embodiment,CTP extends the T_(max) of a coagulation factor. In another embodiment,CTP extends the circulatory half-life of a coagulation factor. In someembodiments, CTP enhances the potency of a coagulation factor.

In another embodiment, provided herein is a method of extending thebiological half-life of a coagulation factor, comprising the step ofattaching one to ten CTPs to the carboxy terminus of the coagulationfactor, thereby extending the biological half-life of the coagulationfactor. In another embodiment, provided herein is a method of extendingthe biological half-life of a coagulation factor, comprising the step ofattaching one to five CTPs to the carboxy terminus of the coagulationfactor, thereby extending the biological half-life of the coagulationfactor. In another embodiment, the present invention provides a methodfor extending the circulatory half-life of a coagulation factor. Inanother embodiment, the present invention provides a method forincreasing the half-life of a coagulation factor. In another embodiment,the present invention provides a method for extending the half-life of acoagulation factor.

Coagulation Factor VII (FVII) is a 444 amino acid glycoprotein (50 KDa)secreted by hepatocytes into the bloodstream as an inactive pro-enzyme.Upon tissue injury and exposure to circulating blood, FVII forms acomplex with Tissue Factor (TF) which is a true receptor protein to FVIIand is expressed by various cells localized in the deeper layers of thevessel wall. The formation of this FVII-TF complex leads to activationof FVII. Activated FVII (FVIIa) initiates the extrinsic coagulationpathway by activating Factor IX and Factor X.

FVII belong to a group of Vitamin K-dependent glycoproteins associatedwith the coagulation system. Besides FVII, this group consists of FactorIX, Factor X, Protein C and prothrombin. These proteins have similardomain organizations and are synthesized as precursors with anN-terminal propeptide followed by a mature amino acid sequence. Thepropeptide contains a docking site for gammacarboxylase which convertsglutamic acids (Glu) into gamma carboxy glutamic acids (Gla). Thisdomain is followed by two epidermal growth factor-like (EGF) domains, aconnecting region (CR) and a C-terminal serine protease domain. Prior tosecretion, FVII propeptide is cleaved forming a 406 amino acid singlechain zymogen FVII glycoprotein. After secretion, the protein can beactivated into a disulfide-linked two chain heterodimer, FVIIa, bycleavage in the CR. The plasma concentration of FVII is 10 nM andapproximately 1% circulates in the active form in healthy individuals.

Factor IX (FIX) is a 415 Amino acid (55 KDa) glycoprotein; it belongs toa group of vitamin K dependent glycoproteins associated with thecoagulation system. FIX has a similar domain organization as factorFVII, Factor X, Protein C and prothrombin that are synthesized asprecursors with an N-terminal propeptide followed by a mature amino acidsequence.

FIX is secreted as a single chain molecule that undergoes complexpost-transcriptional modifications, many of which are critical to itsbiochemical and pharmacokinetic properties. Among all thepost-transcriptional modifications, 12 glutamic acid residues near theamino terminus of FIX that are gamma carboxylated by the vitaminK-dependent gamma carboxylase are the most crucial ones. Carboxylationis required for the interaction of FIX with the phospholipid surfacesand for optimal FIX activity. The amino terminus propeptide serves as arecognition site for the gamma carboxylase and thus, following gammacarboxylation, it is cleaved off by the Golgi apparatus serine proteaseknown as Paired basic Amino acid Cleave Enzyme (PACE/Furin). Fouradditional post-transcriptional modifications might occur at the Golgiapparatus: sulfation of tyrosine 155, phosphorylation of serine 158,O-glycosylation on Ser 63 and on 61 and finally, N-glycosylation on Asn157 and 16, but were shown not to be necessary for proper activity ofFIX.

FIX circulates in the plasma (average concentration of 5 μg/ml) as asingle chain inactive zymogen. Upon proteolytic cleavage at two peptidebonds: Arg 145 and Arg 180 by either one or two physiologicalactivators, FVIIa-TF complex or FIXa, the activation peptide is removed,converting FIX to a fully active enzyme consisting of a light and heavychain held together by a single disulfide bond. The N-terminal lightchain contains the non-catalytic gamma carboxyglutamic acid (Gla) andtwo epidermal growth factor-like domains, while the C-terminal heavychain contains the trypsin-like catalytic domain of the molecule. FIXaalone is characterized by poor catalytic activity. However whencomplexed with FVIII, its proteolytic activity increase by 4-5 orders ofmagnitude towards its natural substrate FX.

In another embodiment, provided herein is a method of extending thebiological half-life or a method of improving the area under the curve(AUC) of a coagulation factor, comprising the step of attaching one toten CTPs to the carboxy terminus of the coagulation factor, therebyextending the biological half-life or improving the AUC of thecoagulation factor. In another embodiment, provided herein is a methodof extending the biological half-life or a method of improving the areaunder the curve (AUC) of a coagulation factor, comprising the step ofattaching one to five CTPs to the carboxy terminus of the coagulationfactor, thereby extending the biological half-life or improving the AUCof the coagulation factor. In another embodiment, provided herein is amethod of extending the biological half-life or a method of improvingthe area under the curve (AUC) of FIX, comprising the step of attachingone to five CTPs to the carboxy terminus of the FIX, thereby extendingthe biological half-life or improving the AUC of the FIX. In anotherembodiment, provided herein is a method of extending the biologicalhalf-life or a method of improving the area under the curve (AUC) ofFVII or FVIIa, comprising the step of attaching one to five CTPs to thecarboxy terminus of FVII or FVIIa, thereby extending the biologicalhalf-life or improving the AUC of FVII or FVIIa.

In another embodiment, the present invention provides a method ofextending the biological half-life of a Factor IX (FIX) polypeptide,comprising the step of attaching three chorionic gonadotropin carboxyterminal peptides (CTPs) to the carboxy terminus of said FIXpolypeptide, thereby extending the biological half-life of said FIXpolypeptide. In another embodiment, the present invention furtherprovides a method of extending the biological half-life of a Factor VIIa(FVIIa) polypeptide, comprising the step of attaching up to fivechorionic gonadotropin carboxy terminal peptides (CTPs) to the carboxyterminus of said FVIIa polypeptide, thereby extending the biologicalhalf-life of said FVIIa polypeptide. In one embodiment, three chorionicgonadotropin carboxy terminal peptides (CTPs) are attached to thecarboxy terminus of said FVIIa polypeptide.

In another embodiment, four chorionic gonadotropin carboxy terminalpeptides (CTPs) are attached to the carboxy terminus of said FVIIapolypeptide. In another embodiment, five chorionic gonadotropin carboxyterminal peptides (CTPs) are attached to the carboxy terminus of saidFVIIa polypeptide.

In another embodiment, the present invention provides a method ofimproving the area under the curve (AUC) of a Factor IX (FIX)polypeptide, comprising the step of attaching three chorionicgonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus ofsaid FIX polypeptide, thereby improving the AUC of said FIX polypeptide.In another embodiment, the present invention provides a method ofimproving the area under the curve (AUC) of a Factor VIIa (FVIIa)polypeptide, comprising the step of attaching up to five chorionicgonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus ofsaid FVIIa polypeptide, thereby improving the AUC of said FVIIapolypeptide. In one embodiment, three chorionic gonadotropin carboxyterminal peptides (CTPs) are attached to the carboxy terminus of saidFVIIa polypeptide. In another embodiment, four chorionic gonadotropincarboxy terminal peptides (CTPs) are attached to the carboxy terminus ofsaid FVIIa polypeptide. In another embodiment, five chorionicgonadotropin carboxy terminal peptides (CTPs) are attached to thecarboxy terminus of said FVIIa polypeptide.

In another embodiment, a coagulation factor of the invention is aprotein. In another embodiment, a coagulation factor of the invention isa peptide. In another embodiment, a coagulation factor of the inventionis a polypeptide. In another embodiment, the coagulation factor is anenzyme. In another embodiment, the coagulation factor is a serineprotease. In another embodiment, the coagulation factor is aglycoprotein. In another embodiment, the coagulation factor is atransglutaminase. In another embodiment, the coagulation factor is aninactive zymogen. In another embodiment, the coagulation factor is anycoagulation factor known to one of skill in the art.

In another embodiment, the coagulation factor is Factor V111 (FVIII). Inanother embodiment, the coagulation factor is Factor V (FV). In anotherembodiment, the coagulation factor is Factor XIII (FXIII). In anotherembodiment, the coagulation factor is Factor X (FX). In anotherembodiment, the coagulation factor is fibrin.

In another embodiment, the coagulation factor is Factor VIIa (FVIIa). Inanother embodiment, the coagulation factor is Factor VII (FVII). Inanother embodiment, the coagulation factor is Factor IX (FIX). Inanother embodiment, the coagulation factor is Factor X (FX). In anotherembodiment, the coagulation factor is Factor XIa (FXIa). In anotherembodiment, the coagulation factor is Factor XII (FXII). In anotherembodiment, the coagulation factor is Factor Xa (FXa). In anotherembodiment, the coagulation factor is Factor Va (FVa). In anotherembodiment, the coagulation factor is prothrombin. In anotherembodiment, the coagulation factor is thrombin. In another embodiment,the coagulation factor is Factor XI (FXI). In another embodiment, thecoagulation factor is Von Willebrand factor (vWF). In anotherembodiment, the coagulation factor is Factor VIIIa (FVIIIa). In anotherembodiment, the coagulation factor is B-deleted Domain FVIII (FVIIIBDD).In another embodiment, the coagulation factor is B domain-deleted FVIII(FVIIIBDD). In another embodiment, the coagulation factor is Betadomain-deleted FVIII (FVIIIBDD). In another embodiment, the coagulationfactor is Factor IXa (FIXa). In another embodiment, the coagulationfactor is prekallikrein. In another embodiment, the coagulation factoris kallikrein. In another embodiment, the coagulation factor is FactorXIIa (FXIIa). In another embodiment, the coagulation factor isFibrinogen. In another embodiment, the coagulation factor isthrombomodulin. In another embodiment, the coagulation factor is FactorII (FII).

In another embodiment, the coagulation factor is a glycoprotein. Inanother embodiment, the coagulation factor is a vitamin K-dependentglycoprotein. In another embodiment, the coagulation factor is a vitaminK-independent glycoprotein.

In another embodiment, the coagulation factor is a recombinant protein.In another embodiment, the coagulation factor is a recombinantglycoprotein. In another embodiment, the coagulation factor is arecombinant glycoprotein FV. In another embodiment, the coagulationfactor is a recombinant FVI. In another embodiment, the coagulationfactor is a recombinant FVII. In another embodiment, the coagulationfactor is a recombinant FVIII. In another embodiment, the coagulationfactor is a recombinant FIX. In another embodiment, the coagulationfactor is a recombinant FX. In another embodiment, the coagulationfactor is a recombinant FXI. In another embodiment, the coagulationfactor is a recombinant FXII. In another embodiment, the coagulationfactor is a recombinant FvW. In another embodiment, the coagulationfactor is a recombinant FII. In another embodiment, the coagulationfactor is a recombinant FIXa. In another embodiment, the coagulationfactor is a recombinant FXIa. In another embodiment, the coagulationfactor is a recombinant fibrin. In another embodiment, the coagulationfactor is a recombinant FVIIa. In another embodiment, the coagulationfactor is a recombinant FXa. In another embodiment, the coagulationfactor is a recombinant FVa. In another embodiment, the coagulationfactor is a recombinant prothrombin. In another embodiment, thecoagulation factor is a recombinant thrombin. In another embodiment, thecoagulation factor is a recombinant FVIIIa. In another embodiment, thecoagulation factor is a recombinant prekallikrein. In anotherembodiment, the coagulation factor is a recombinant kallikrein. Inanother embodiment, the coagulation factor is a recombinant FXIIa. Inanother embodiment, the coagulation factor is any known recombinantcoagulation factor. In another embodiment, the coagulation factorcomprising a signal peptide is any known recombinant coagulation factor.

In another embodiment, a coagulation factor comprises 1-10 CTP repeatsattached to the C-terminus and no CTPs attached to the N-terminus. Inanother embodiment, a coagulation factor comprises at least one CTPattached to the C-terminus and no CTPs attached to the N-terminus. Inanother embodiment, a coagulation factor comprising 1-10 CTP repeatsattached to the C-terminus and no CTPs attached to the N-terminus is anengineered coagulation factor. In another embodiment, a coagulationfactor comprising at least one CTP attached to the C-terminus and noCTPs attached to the N-terminus is an engineered coagulation factor. Inanother embodiment, a coagulation factor comprising 1-10 CTP repeatsattached to the C-terminus and no CTPs attached to the N-terminus is aconjugated coagulation factor. In another embodiment, a coagulationfactor comprising at least one CTP attached to the C-terminus and noCTPs attached to the N-terminus is a conjugated coagulation factor.

In one embodiment, the present invention provides a CTP-modified FactorIX (FIX) polypeptide consisting of a FIX polypeptide and threegonadotropin carboxy terminal peptides (CTPs) attached to the carboxyterminus of said CTP-modified FIX polypeptide.

In another embodiment, the present invention further provides aCTP-modified Factor VIIa (FVIIa) polypeptide consisting of a FVIIapolypeptide and five gonadotropin carboxy terminal peptides (CTPs)attached to the carboxy terminus of said FVIIa.

In another embodiment, the coagulation factor is a coagulation factorcomprising a domain organization similar or identical to the domainorganization of FIX, FVII, Factor X, Protein C, or prothrombin. Inanother embodiment, the coagulation factor is synthesized as a precursorwith an N-terminal propeptide. In another embodiment, the coagulationfactor as used herein is in an inactive pro-enzyme form. In anotherembodiment, the coagulation factor is produced in hepatocytes. Inanother embodiment, the coagulation factor comprises a docking site forgammacarboxylase which converts glutamic acids (Glu) into gamma carboxyglutamic acids (Gla). In another embodiment, the coagulation factor asused herein is a commercially available coagulation factor.

In one embodiment, the nucleic acid sequence encoding Factor VIIcomprises the following nucleic acid sequence:

(SEQ ID NO: 11) ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcagtcttcgtaacccaggaggaagcccacggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgcggccgggctccctggagagggagtgcaaggaggagcagtgctccttcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatggggaccagtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccctgccttcgagggccggaactgtgagacgcacaaggatgaccagctgatctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaagcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacctattctagaaaaaagaaatgccagcaaaccccaaggccgaattgtggggggcaaggtgtgccccaaaggggagtgtccatggcaggtcctgttgttggtgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactgtttcgacaaaatcaagaactggaggaacctgatcgcggtgctgggcgagcacgacctcagcgagcacgacggggatgagcagagccggcgggtggcgcaggtcatcatccccagcacgtacgtcccgggcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtggtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagcggctggggccagctgctggaccgtggcgccacggccctggagctcatggtcctcaacgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtgggagactccccaaatatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggcccacatgccacccactaccggggcacgtggtacctgacgggcatcgtcagctggggccagggctgcgcaaccgtgggccactttggggtgtacaccagggtctcccagtacatcgagtggctgcaaaagctcatgcgctcagagccacgcccaggagtcctcctgcgagccccatttccctgaggatgcggccgc.

In another embodiment, the amino acid sequence of Factor VII comprisesthe following amino acid sequence:

(SEQ ID NO: 9) MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRA PFP.

In another embodiment, the amino acid sequence of Factor VII comprisesthe following amino acid sequence:

(SEQ ID NO: 10) MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRA PFP*GCGR.

In another embodiment, the nucleic acid sequence encoding Factor VII-CTP(attached to the carboxy terminus) comprises the following nucleic acidsequence:

(SEQ ID NO: 12) ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcagtcttcgtaacccaggaggaagcccacggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgcggccgggctccctggagagggagtgcaaggaggagcagtgctccttcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatggggaccagtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccctgccttcgagggccggaactgtgagacgcacaaggatgaccagctgatctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaagcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacctattctagaaaaaagaaatgccagcaaaccccaaggccgaattgtggggggcaaggtgtgccccaaaggggagtgtccatggcaggtcctgttgttggtgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactgtttcgacaaaatcaagaactggaggaacctgatcgcggtgctgggcgagcacgacctcagcgagcacgacggggatgagcagagccggcgggtggcgcaggtcatcatccccagcacgtacgtcccgggcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtggtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagcggctggggccagctgctggaccgtggcgccacggccctggagctcatggtcctcaacgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtgggagactccccaaatatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggcccacatgccacccactaccggggcacgtggtacctgaccggcatcgtgagctggggccagggctgcgccaccgtgggccacttcggcgtgtacaccagggtgtcccagtacatcgagtggctgcagaaactgatgagaagcgagcccagacccggcgtgctgctgagagcccccttccccagcagcagctccaaggcccctccccctagcctgcccagccctagcagactgcctgggcccagcgacacccccatcctgcc ccagtgaggatccgcggccgc.

In another embodiment, the amino acid sequence of Factor VII-CTP(attached to the carboxy terminus) comprises the following amino acidsequence:

(SEQ ID NO: 13) MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRTVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTTWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQ*.

In another embodiment, the nucleic acid sequence encoding FactorVII-CTP-CTP (attached to the carboxy terminus) comprises the followingnucleic acid sequence:

(SEQ ID NO: 14) ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcagtcttcgtaacccaggaggaagcccacggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgcggccgggctccctggagagggagtgcaaggaggagcagtgctccttcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatggggaccagtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccctgccttcgagggccggaactgtgagacgcacaaggatgaccagctgatctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaagcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacctattctagaaaaaagaaatgccagcaaaccccaaggccgaattgtggggggcaaggtgtgccccaaaggggagtgtccatggcaggtcctgttgttggtgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactgtttcgacaaaatcaagaactggaggaacctgatcgcggtgctgggcgagcacgacctcagcgagcacgacggggatgagcagagccggcgggtggcgcaggtcatcatccccagcacgtacgtcccgggcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtggtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagcggctggggccagctgctggaccgtggcgccacggccctggagctcatggtcctcaacgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtgggagactccccaaatatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggcccacatgccacccactaccggggcacgtggtacctgaccggcatcgtgagctggggccagggctgcgccaccgtgggccacttcggcgtgtacaccagggtgtcccagtacatcgagtggctgcagaaactgatgagaagcgagcccagacccggcgtgctgctgagagcccccttccccagcagcagctccaaggcccctccccctagcctgcccagccctagcagactgcctgggccctccgacacaccaatcctgccacagagcagctcctctaaggcccctcctccatccctgccatccccctcccggctgccaggcccctctgacacccctatcctgcctcagtgatgaaggt ctggatccgcggccgc.

In another embodiment, the amino acid sequence of Factor VII-CTP-CTP(attached to the carboxy terminus) comprises the following amino acidsequence:

(SEQ ID NO: 15) MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRTVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTTWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLP GPSDTPILPQ**.

In another embodiment, the nucleic acid sequence encoding FactorVII-CTP-CTP-CTP (attached to the carboxy terminus) comprises thefollowing nucleic acid sequence:

(SEQ ID NO: 24) ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcagtcttcgtaacccaggaggaagcccacggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgcggccgggctccctggagagggagtgcaaggaggagcagtgctccttcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatggggaccagtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccctgccttcgagggccggaactgtgagacgcacaaggatgaccagctgatctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaagcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacctattctagaaaaaagaaatgccagcaaaccccaaggccgaattgtggggggcaaggtgtgccccaaaggggagtgtccatggcaggtcctgttgttggtgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactgtttcgacaaaatcaagaactggaggaacctgatcgcggtgctgggcgagcacgacctcagcgagcacgacggggatgagcagagccggcgggtggcgcaggtcatcatccccagcacgtacgtcccgggcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtggtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagcggctggggccagctgctggaccgtggcgccacggccctggagctcatggtcctcaacgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtgggagactccccaaatatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggcccacatgccacccactaccggggcacgtggtacctgaccggcatcgtgagctggggccagggctgcgccaccgtgggccacttcggcgtgtacaccagggtgtcccagtacatcgagtggctgcagaaactgatgagaagcgagcccagacccggcgtgctgctgagagcccccttccccagcagcagctccaaggcccctccccctagcctgcccagccctagcagactgcctgggcccagtgacacccctatcctgcctcagtccagctccagcaaggccccaccccctagcctgccttctccttctcggctgcctggccccagcgatactccaattctgccccagtcctccagcagtaaggctccccctccatctctgccatcccccagcagactgccaggcccttctgatacacccatcctcccacagtgatgaggatccgcggccgcttaa ttaa.

In another embodiment, the amino acid sequence of Factor VII-CTP-CTP-CTP(attached to the carboxy terminus) comprises the following amino acidsequence:

(SEQ ID NO: 25) MVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRTVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTTWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ**.

In another embodiment, the nucleic acid sequence encoding FactorVII-(CTP)₄ (attached to the carboxy terminus) comprises the followingnucleic acid sequence:

(SEQ ID NO: 26) ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcagtcttcgtaacccaggaggaagcccacggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgcggccgggctccctggagagggagtgcaaggaggagcagtgctccttcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatggggaccagtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccctgccttcgagggccggaactgtgagacgcacaaggatgaccagctgatctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaagcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacctattctagaaaaaagaaatgccagcaaaccccaaggccgaattgtggggggcaaggtgtgccccaaaggggagtgtccatggcaggtcctgttgttggtgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactgtttcgacaaaatcaagaactggaggaacctgatcgcggtgctgggcgagcacgacctcagcgagcacgacggggatgagcagagccggcgggtggcgcaggtcatcatccccagcacgtacgtcccgggcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtggtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagcggctggggccagctgctggaccgtggcgccacggccctggagctcatggtcctcaacgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtgggagactccccaaatatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggcccacatgccacccactaccggggcacgtggtacctgaccggcatcgtgagctggggccagggctgcgccaccgtgggccacttcggcgtgtacaccagggtgtcccagtacatcgagtggctgcagaaactgatgagaagcgagcccagacccggcgtgctgctgagagcccccttccccagcagcagctccaaggcccctccccctagcctgcccagccctagcagactgcctgggcccagtgacacccctatcctgcctcagtccagctccagcaaggccccaccccctagcctgccttctccttctcggctgcctggccccagcgatactccaattctgccccagtcctccagcagtaaggctccccctccatctctgccatcccccagcagactgccaggcccttctgatacacccatcctcccacagtgatgaggatccgc.

In another embodiment, the amino acid sequence of Factor VII-(CTP)₄(attached to the carboxy terminus) comprises the following amino acidsequence:

(SEQ ID NO: 27) LEDMVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ**G.

In another embodiment, the nucleic acid sequence encoding FactorVII-(CTP)₅ (attached to the carboxy terminus) comprises the followingnucleic acid sequence:

(SEQ ID NO: 28) ctcgaggacatggtctcccaggccctcaggctcctctgccttctgcttgggcttcagggctgcctggctgcagtcttcgtaacccaggaggaagcccacggcgtcctgcaccggcgccggcgcgccaacgcgttcctggaggagctgcggccgggctccctggagagggagtgcaaggaggagcagtgctccttcgaggaggcccgggagatcttcaaggacgcggagaggacgaagctgttctggatttcttacagtgatggggaccagtgtgcctcaagtccatgccagaatgggggctcctgcaaggaccagctccagtcctatatctgcttctgcctccctgccttcgagggccggaactgtgagacgcacaaggatgaccagctgatctgtgtgaacgagaacggcggctgtgagcagtactgcagtgaccacacgggcaccaagcgctcctgtcggtgccacgaggggtactctctgctggcagacggggtgtcctgcacacccacagttgaatatccatgtggaaaaatacctattctagaaaaaagaaatgccagcaaaccccaaggccgaattgtggggggcaaggtgtgccccaaaggggagtgtccatggcaggtcctgttgttggtgaatggagctcagttgtgtggggggaccctgatcaacaccatctgggtggtctccgcggcccactgtttcgacaaaatcaagaactggaggaacctgatcgcggtgctgggcgagcacgacctcagcgagcacgacggggatgagcagagccggcgggtggcgcaggtcatcatccccagcacgtacgtcccgggcaccaccaaccacgacatcgcgctgctccgcctgcaccagcccgtggtcctcactgaccatgtggtgcccctctgcctgcccgaacggacgttctctgagaggacgctggccttcgtgcgcttctcattggtcagcggctggggccagctgctggaccgtggcgccacggccctggagctcatggtcctcaacgtgccccggctgatgacccaggactgcctgcagcagtcacggaaggtgggagactccccaaatatcacggagtacatgttctgtgccggctactcggatggcagcaaggactcctgcaagggggacagtggaggcccacatgccacccactaccggggcacgtggtacctgaccggcatcgtgagctggggccagggctgcgccaccgtgggccacttcggcgtgtacaccagggtgtcccagtacatcgagtggctgcagaaactgatgagaagcgagcccagacccggcgtgctgctgagagcccccttccccagcagcagctccaaggcccctccccctagcctgcccagccctagcagactgcctgggccctctgacacccctatcctgcctcagtccagctcctctaaggctccaccaccttccctgcctagcccttcaagactgccaggccctagcgatacaccaattctgccccagtcctccagcagcaaggctcccccacctagcctgccttctccatcaaggctgcctggcccatccgataccccaattttgcctcagagcagctctagcaaggcacctccccccagtctgccctctccaagcagactccctggcccttcagacactccaatcctcccacagtcctctagctctaaagctccacctcccagcctgcccagccctagtagactccccggaccttctgatacccccatcttgccccagtga tgaggatccgc.

In another embodiment, the amino acid sequence of Factor VII-(CTP)₅(attached to the carboxy terminus) comprises the following amino acidsequence:

(SEQ ID NO: 29) LEDMVSQALRLLCLLLGLQGCLAAVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILP Q**GS.

In another embodiment, the nucleic acid sequence encoding Factor IXcomprises the following nucleic acid sequence:

(SEQ ID NO: 16) gcgatcgccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccattgccttttaggatatctactcagtgctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaaccttgagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaaggaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttctaagctcacccgtgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactcaaagcacccaatcatttaatgacttcactcgagttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgcattctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacacctatttgcattgctgacaaggaatacacgaacatcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttctccagtaccttagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatgaaaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaacaaagctcacttgaacgcggccgc.

In another embodiment, the amino acid sequence of Factor IX comprisesthe following amino acid sequence:

(SEQ ID NO: 17) MQRVNMPMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKY GIYTKVSRYVNWIKEKTKLT*.

In another embodiment, the nucleic acid sequence encoding Factor IX-CTP(attached to the carboxy terminus) comprises the following nucleic acidsequence:

(SEQ ID NO: 18) gcgatcgccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttaggatatctactcagtgctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaaccttgagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaaggaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttctaagctcacccgtgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactcaaagcacccaatcatttaatgacttcactcgagttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgcattctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacacctatttgcattgctgacaaggaatacacgaacatcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtaccttagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatgaaaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaacaaagctcactagctccagcagcaaggcccctcccccgagcctgccctccccaagcaggctgcctgggccctccgacacaccaatcctgccacagtgatgaaggtctggatccgcggcc gc.

In another embodiment, the amino acid sequence of Factor IX-CTP(attached to the carboxy terminus) comprises the following amino acidsequence:

(SEQ ID NO: 19) MQRVNMPMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAPPPSLPSPSRLPGPSDTPILPQ**.

In another embodiment, the nucleic acid sequence encoding FactorIX-CTP-CTP (attached to the carboxy terminus) comprises the followingnucleic acid sequence:

(SEQ ID NO: 20) gcgatcgccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttaggatatctactcagtgctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaaccttgagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaaggaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttctaagctcacccgtgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactcaaagcacccaatcatttaatgacttcactcgagttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgcattctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacacctatttgcattgctacaaggaatacacgaacatcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtaccttagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatgaaaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaacaaagctcactagctccagcagcaaggcccctcccccgagcctgccctccccaagcaggctgcctgggccctccgacacaccaatcctgccacagagcagctcctctaaggcccctcctccatccctgccatccccctcccggctgcctggcccctctgacacccctatcctgcctcagtgatgaaggtctggatccgcggccgc.

In another embodiment, the amino acid sequence of Factor IX-CTP-CTP(attached to the carboxy terminus) comprises the following amino acidsequence:

(SEQ ID NO: 21) MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSL PSPSRLPGPSDTPILPQ**.

In another embodiment, the nucleic acid sequence encoding FactorIX-(CTP)₃ (attached to the carboxy terminus) comprises the followingnucleic acid sequence:

(SEQ ID NO: 30) tctagagtcgaccccgccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttaggatatctactcagtgctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaaccttgagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaaggaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttctaagctcacccgtgctgaggcagtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactcaaagcacccaatcatttaatgacttcactcgagttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgcattctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacacctatttgcattgctgacaaggaatacacgaacatcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtaccttagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatgaaaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaacaaagctcactagctccagcagcaaggcccctcccccgagcctgccctccccaagcaggctgcctgggcccagtgacacccctatcctgcctcagtccagctccagcaaggccccaccccctagcctgccttctccttctcggctgcctggccccagcgatactccaattctgccccagtcctccagcagtaaggctccccctccatctctgccatcccccagcagactgccaggcccttctgatacacccatcctcccacagtgatgaggatccgcggccgc.

In another embodiment, the amino acid sequence of Factor IX-(CTP)₃(attached to the carboxy terminus) comprises the following amino acidsequence:

(SEQ ID NO: 31) MQRVNMPMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ**.

In another embodiment, the nucleic acid sequence encoding FactorIX-(CTP)₄ (attached to the carboxy terminus) comprises the followingnucleic acid sequence:

(SEQ ID NO: 32) tctagagtcgaccccgccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttaggatatctactcagtgctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaaccttgagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaaggaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttctaagctcacccgtgctgaggcagtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactcaaagcacccaatcatttaatgacttcactcgagttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgcattctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacacctatttgcattgctgacaaggaatacacgaacatcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtaccttagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatgaaaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaacaaagctcactagctccagcagcaaggcccctcccccgagcctgccctccccaagcaggctgcctgggccctctgacacccctatcctgcctcagtccagctcctctaaggccccaccaccttccctgcctagcccttcaagactgccaggccctagcgatacaccaattctgccccagtcctccagcagcaaggctcccccacctagcctgccttctccatcaaggctgcctggcccatccgataccccaattttgcctcagagcagctctagcaaggcacctccccccagtctgccctctccaagcagactccctggcccttcagacactcccattctgccacagtgatgaggatccg cggccgc.

In another embodiment, the amino acid sequence of Factor IX-(CTP)₄(attached to the carboxy terminus) comprises the following amino acidsequence:

(SEQ ID NO: 33) SRVDPAMQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQ**GSAA.

In another embodiment, the nucleic acid sequence encoding FactorIX-(CTP)₅ (attached to the carboxy terminus) comprises the followingnucleic acid sequence:

(SEQ ID NO: 34) ctagagtcgaccccgccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttaggatatctactcagtgctgaatgtacagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaaccttgagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaaggaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttctaagctcacccgtgctgaggcagtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactcaaagcacccaatcatttaatgacttcactcgagttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgcattctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacacctatttgcattgctgacaaggaatacacgaacatcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtaccttagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatgaaaggcaaatatggaatatataccaaggtatcccggtatgtcaactggattaaggaaaaaacaaagctcactagctccagcagcaaggcccctcccccgagcctgccctccccaagcaggctgcctgggccctctgacacccctatcctgcctcagtccagctcctctaaggctccaccaccttccctgcctagcccttcaagactgccaggccctagcgatacaccaattctgccccagtcctccagcagcaaggctcccccacctagcctgccttctccatcaaggctgcctggcccatccgataccccaattttgcctcagagcagctctagcaaggcacctccccccagtctgccctctccaagcagactccctggcccttcagacactccaatcctcccacagtcctctagctctaaagctccacctcccagcctgcccagccctagtagactccccggaccttctgatacccccatcttgccccagtgatgaggatccgcggccgc.

In another embodiment, the amino acid sequence of Factor IX-(CTP)₅(attached to the carboxy terminus) comprises the following amino acidsequence:

(SEQ ID NO: 35) RVDPAMQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSDTPILPQSSSSKAPPPSLPSPSRLPGPSD TPILPQ**GSAA.

In another embodiment, furin is added to a cell expressing thecoagulation factor-CTP of the invention. In another embodiment, furinincreases the production efficiency of a coagulation factor-CTP of theinvention in a cell. In another embodiment, furin is co-transfected withthe vector comprising the coding sequence of the coagulation factor-CTPof the invention. In another embodiment, furin is encoded by a separatevector. In another embodiment, furin and a coagulation factor-CTP areencoded by one vector. In another embodiment, the coding sequence offurin is inserted into pCI-DHFR. In another embodiment, the codingsequence of furin is engineered in pCI-dhfr/smal+NotI, Furin/AsislF.I.+NotI.

In another embodiment, the nucleic acid sequence encoding furincomprises the following nucleic acid sequence:

(SEQ ID NO: 22) tctagagtcgaccccgccatggagctgaggccctggttgctatgggtggtagcagcaacaggaaccttggtcctgctagcagctgatgctcagggccagaaggtcttcaccaacacgtgggctgtgcgcatccctggaggcccagcggtggccaacagtgtggcacggaagcatgggttcctcaacctgggccagatcttcggggactattaccacttctggcatcgaggagtgacgaagcggtccctgtcgcctcaccgcccgcggcacagccggctgcagagggagcctcaagtacagtggctggaacagcaggtggcaaagcgacggactaaacgggacgtgtaccaggagcccacagaccccaagtttcctcagcagtggtacctgtctggtgtcactcagcgggacctgaatgtgaaggcggcctgggcgcagggctacacagggcacggcattgtggtctccattctggacgatggcatcgagaagaaccacccggacttggcaggcaattatgatcctggggccagttttgatgtcaatgaccaggaccctgacccccagcctcggtacacacagatgaatgacaacaggcacggcacacggtgtgcgggggaagtggctgcggtggccaacaacggtgtctgtggtgtaggtgtggcctacaacgcccgcattggaggggtgcgcatgctggatggcgaggtgacagatgcagtggaggcacgctcgctgggcctgaaccccaaccacatccacatctacagtgccagctggggccccgaggatgacggcaagacagtggatgggccagcccgcctcgccgaggaggccttcttccgtggggttagccagggccgaggggggctgggctccatctttgtctgggcctcggggaacgggggccgggaacatgacagctgcaactgcgacggctacaccaacagtatctacacgctgtccatcagcagcgccacgcagtttggcaacgtgccgtggtacagcgaggcctgctcgtccacactggccacgacctacagcagtggcaaccagaatgagaagcagatcgtgacgactgacttgcggcagaagtgcacggagtctcacacgggcacctcagcctctgcccccttagcagccggcatcattgctctcaccctggaggccaataagaacctcacatggcgggacatgcaacacctggtggtacagacctcgaagccagcccacctcaatgccaacgactgggccaccaatggtgtgggccggaaagtgagccactcatatggctacgggcttttggacgcaggcgccatggtggccctggcccagaattggaccacagtggccccccagcggaagtgcatcatcgacatcctcaccgagcccaaagacatcgggaaacggctcgaggtgcggaagaccgtgaccgcgtgcctgggcgagcccaaccacatcactcggctggagcacgctcaggcgcggctcaccctgtcctataatcgccgtggcgacctggccatccacctggtcagccccatgggcacccgctccaccctgctggcagccaggccacatgactactccgcagatgggtttaatgactgggccttcatgacaactcattcctgggatgaggatccctctggcgagtgggtcctagagattgaaaacaccagcgaagccaacaactatgggacgctgaccaagttcaccctcgtactctatggcaccgcccctgaggggctgcccgtacctccagaaagcagtggctgcaagaccctcacgtccagtcaggcctgtgtggtgtgcgaggaaggcttctccctgcaccagaagagctgtgtccagcactgccctccaggcttcgccccccaagtcctcgatacgcactatagcaccgagaatgacgtggagaccatccgggccagcgtctgcgccccctgccacgcctcatgtgccacatgccaggggccggccctgacagactgcctcagctgccccagccacgcctccttggaccctgtggagcagacttgctcccggcaaagccagagcagccgagagtccccgccacagcagcagccacctcggctgcccccggaggtggaggcggggcaacggctgcgggcagggctgctgccctcacacctgcctgaggtggtggccggcctcagctgcgccttcatcgtgctggtcttcgtcactgtcttcctggtcctgcagctgcgctctggctttagttttcggggggtgaaggtgtacaccatggaccgtggcctcatctcctacaaggggctgccccctgaagcctggcaggaggagtgcccgtctgactcagaagaggacgagggccggggcgagaggaccgcctttatcaaagaccagagcgccctc tgaacgcggccgc.

In another embodiment, the amino acid sequence of furin comprises thefollowing amino acid sequence:

(SEQ ID NO: 23) MELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARKHGFLNLGQIFGDYYHFWHRGVTKRSLSPHRPRHSRLQREPQVQWLEQQVAKRRTKRDVYQEPTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIVVSILDDGIEKNHPDLAGNYDPGASFDVNDQDPDPQPRYTQMNDNRHGTRCAGEVAAVANNGVCGVGVAYNARIGGVRMLDGEVTDAVEARSLGLNPNHIHIYSASWGPEDDGKTVDGPARLAEEAFFRGVSQGRGGLGSIFVWASGNGGREHDSCNCDGYTNSIYTLSISSATQFGNVPWYSEACSSTLATTYSSGNQNEKQIVTTDLRQKCTESHTGTSASAPLAAGIIALTLEANKNLTWRDMQHLVVQTSKPAHLNANDWATNGVGRKVSHSYGYGLLDAGAMVALAQNWTTVAPQRKCIIDILTEPKDIGKRLEVRKTVTACLGEPNHITRLEHAQARLTLSYNRRGDLAIHLVSPMGTRSTLLAARPHDYSADGFNDWAFMTTHSWDEDPSGEWVLEIENTSEANNYGTLTKFTLVLYGTAPEGLPVPPESSGCKTLTSSQACVVCEEGFSLHQKSCVQHCPPGFAPQVLDTHYSTENDVETIRASVCAPCHASCATCQGPALTDCLSCPSHASLDPVEQTCSRQSQSSRESPPQQQPPRLPPEVEAGQRLRAGLLPSHLPEVVAGLSCAFIVLVFVTVFLVLQLRSGFSFRGVKVYTMDRGLISYKGLPPEAWQEECPSDSEEDEGRGERTAFIKDQSAL*.

In one embodiment, the term coagulation factor further includes ahomologue of a known coagulation factor. In one embodiment, thehomologue has a coagulating activity. In some embodiments, homologyaccording to the present invention also encompasses deletion, insertion,or substitution variants, including an amino acid substitution, thereofand biologically active polypeptide fragments thereof. In oneembodiment, the variant comprises conservative substitutions, ordeletions, insertions, or substitutions that do not significantly alterthe three dimensional structure of the coagulation factor. In anotherembodiment, the deletion, insertion, or substitution does not alter thefunction of interest of the coagulation factor, which in one embodiment,is binding to a particular binding partner.

In another embodiment, the invention includes a homologue of acoagulation factor. In another embodiment, the invention includes ahomologue of a coagulation factor having a coagulation activity. Inanother embodiment, the invention includes a homologue of a coagulationfactor having functional binding. In another embodiment, the inventionincludes homologues of a coagulation factor as described herein having acoagulation activity. In another embodiment, the invention includeshomologues of a coagulation factor as described herein having functionalbinding. In another embodiment, homologues e.g., polypeptides which areat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 87%, at least 89%, atleast 91%, at least 93%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% homologous to a coagulation factor asdetermined using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters.

In another embodiment, the invention includes homologues of furin. Inanother embodiment, the invention includes homologues of furinmaintaining a function of interest, which in one embodiment is cleavingof a precursor protein. In another embodiment, homologues e.g.,polypeptides which are at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 87%, at least 89%, at least 91%, at least 93%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% homologous to afurin as determined using BlastP software of the National Center ofBiotechnology Information (NCBI) using default parameters.

In another embodiment, provided herein is a polypeptide comprising acoagulation factor and one to ten gonadotropin carboxy terminal peptides(CTPs) attached to the carboxy terminus of the coagulation factor. Inanother embodiment, provided herein is a polypeptide comprising acoagulation factor and one to three gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptidecomprising a coagulation factor and one to five gonadotropin carboxyterminal peptides (CTPs) attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide comprising a coagulation factor having at least one CTP onits carboxy terminus.

In another embodiment, provided herein is a polypeptide consisting of acoagulation factor and one to five gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of the coagulationfactor.

In another embodiment, provided herein is a polypeptide consistingessentially of a coagulation factor and one to five CTPs attached to thecarboxy terminus of the coagulation factor.

It is to be understood that the compositions and methods of the presentinvention comprising the elements or steps as described herein may, inanother embodiment, consist of those elements or steps, or in anotherembodiment, consist essentially of those elements or steps. In someembodiments, the term “comprise” refers to the inclusion of theindicated active agent, such as the CTP-modified coagulation factor, aswell as inclusion of other active agents, and pharmaceuticallyacceptable carriers, excipients, emollients, stabilizers, etc., as areknown in the pharmaceutical industry. In some embodiments, the term“consisting essentially of” refers to a composition, whose only activeingredient is the indicated active ingredient, however, other compoundsmay be included which are for stabilizing, preserving, etc. theformulation, but are not involved directly in the therapeutic effect ofthe indicated active ingredient. In some embodiments, the term“consisting essentially of” may refer to components which facilitate therelease of the active ingredient. In some embodiments, the term“consisting” refers to a composition, which contains the activeingredient and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the present invention provides a polypeptidecomprising a coagulation factor and two gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptidecomprising a coagulation factor and two to three CTPs attached to thecarboxy terminus of the coagulation factor.

In another embodiment, provided herein is a polypeptide comprising acoagulation factor and two to four CTPs attached to the carboxy terminusof the coagulation factor. In another embodiment, provided herein is apolypeptide comprising a coagulation factor and two to five CTPsattached to the carboxy terminus of the coagulation factor. In anotherembodiment, provided herein is a polypeptide comprising a coagulationfactor and two to six CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide comprising a coagulation factor and two to seven CTPsattached to the carboxy terminus of the coagulation factor. In anotherembodiment, provided herein is a polypeptide comprising a coagulationfactor and two to eight CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide comprising a coagulation factor and two to nine CTPsattached to the carboxy terminus of the coagulation factor. In anotherembodiment, provided herein is a polypeptide comprising a coagulationfactor and two to ten CTPs attached to the carboxy terminus of thecoagulation factor.

In one embodiment, the present invention provides a polypeptidecomprising a coagulation factor and three gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptidecomprising a coagulation factor and three to four CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide comprising a coagulation factor andthree to five CTPs attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptidecomprising a coagulation factor and three to six CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide comprising a coagulation factor andthree to seven CTPs attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptidecomprising a coagulation factor and three to eight CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide comprising a coagulation factor andthree to nine CTPs attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptidecomprising a coagulation factor and three to ten CTPs attached to thecarboxy terminus of the coagulation factor.

In one embodiment, the present invention provides a polypeptidecomprising a coagulation factor and four gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptidecomprising a coagulation factor and four to five CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide comprising a coagulation factor andfour to six CTPs attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptidecomprising a coagulation factor and four to seven CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide comprising a coagulation factor andfour to eight CTPs attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptidecomprising a coagulation factor and four to nine CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide comprising a coagulation factor andfour to ten CTPs attached to the carboxy terminus of the coagulationfactor.

In one embodiment, the present invention provides a polypeptidecomprising a coagulation factor and five gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptidecomprising a coagulation factor and five to six CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide comprising a coagulation factor andfive to seven CTPs attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptidecomprising a coagulation factor and five to eight CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide comprising a coagulation factor andfive to nine CTPs attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptidecomprising a coagulation factor and five to ten CTPs attached to thecarboxy terminus of the coagulation factor.

In one embodiment, the present invention provides a polypeptideconsisting of a coagulation factor and two gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptideconsisting of a coagulation factor and two to three CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide consisting of a coagulation factor andtwo to four CTPs attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptideconsisting of a coagulation factor and two to five CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide consisting of a coagulation factor andtwo to six CTPs attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptideconsisting of a coagulation factor and two to seven CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide consisting of a coagulation factor andtwo to eight CTPs attached to the carboxy terminus of the coagulationfactor. In another embodiment, provided herein is a polypeptideconsisting of a coagulation factor and two to nine CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide consisting of a coagulation factor andtwo to ten CTPs attached to the carboxy terminus of the coagulationfactor.

In one embodiment, the present invention provides a polypeptideconsisting of a coagulation factor and three gonadotropin carboxyterminal peptides (CTPs) attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting of a coagulation factor and three to four CTPsattached to the carboxy terminus of the coagulation factor. In anotherembodiment, provided herein is a polypeptide consisting of a coagulationfactor and three to five CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting of a coagulation factor and three to six CTPsattached to the carboxy terminus of the coagulation factor. In anotherembodiment, provided herein is a polypeptide consisting of a coagulationfactor and three to seven CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting of a coagulation factor and three to eight CTPsattached to the carboxy terminus of the coagulation factor. In anotherembodiment, provided herein is a polypeptide consisting of a coagulationfactor and three to nine CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting of a coagulation factor and three to ten CTPsattached to the carboxy terminus of the coagulation factor.

In one embodiment, the present invention provides a polypeptideconsisting of a coagulation factor and four gonadotropin carboxyterminal peptides (CTPs) attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting of a coagulation factor and four to five CTPsattached to the carboxy terminus of the coagulation factor. In anotherembodiment, provided herein is a polypeptide consisting of a coagulationfactor and four to six CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting of a coagulation factor and four to seven CTPsattached to the carboxy terminus of the coagulation factor. In anotherembodiment, provided herein is a polypeptide consisting of a coagulationfactor and four to eight CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting of a coagulation factor and four to nine CTPsattached to the carboxy terminus of the coagulation factor. In anotherembodiment, provided herein is a polypeptide consisting of a coagulationfactor and four to ten CTPs attached to the carboxy terminus of thecoagulation factor.

In one embodiment, the present invention provides a polypeptideconsisting of a coagulation factor and five gonadotropin carboxyterminal peptides (CTPs) attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting of a coagulation factor and five to six CTPsattached to the carboxy terminus of the coagulation factor. In anotherembodiment, provided herein is a polypeptide consisting of a coagulationfactor and five to seven CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting of a coagulation factor and five to eight CTPsattached to the carboxy terminus of the coagulation factor. In anotherembodiment, provided herein is a polypeptide consisting of a coagulationfactor and five to nine CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting of a coagulation factor and five to ten CTPsattached to the carboxy terminus of the coagulation factor.

In one embodiment, the present invention provides a polypeptideconsisting essentially of a coagulation factor and two gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting essentially of a coagulation factor and two tothree CTPs attached to the carboxy terminus of the coagulation factor.In another embodiment, provided herein is a polypeptide consistingessentially of a coagulation factor and two to four CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide consisting essentially of a coagulationfactor and two to five CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting essentially of a coagulation factor and two tosix CTPs attached to the carboxy terminus of the coagulation factor. Inanother embodiment, provided herein is a polypeptide consistingessentially of a coagulation factor and two to seven CTPs attached tothe carboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide consisting essentially of a coagulationfactor and two to eight CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting essentially of a coagulation factor and two tonine CTPs attached to the carboxy terminus of the coagulation factor. Inanother embodiment, provided herein is a polypeptide consistingessentially of a coagulation factor and two to ten CTPs attached to thecarboxy terminus of the coagulation factor.

In one embodiment, the present invention provides a polypeptideconsisting essentially of a coagulation factor and three gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting essentially of a coagulation factor and three tofour CTPs attached to the carboxy terminus of the coagulation factor. Inanother embodiment, provided herein is a polypeptide consistingessentially of a coagulation factor and three to five CTPs attached tothe carboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide consisting essentially of a coagulationfactor and three to six CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting essentially of a coagulation factor and three toseven CTPs attached to the carboxy terminus of the coagulation factor.In another embodiment, provided herein is a polypeptide consistingessentially of a coagulation factor and three to eight CTPs attached tothe carboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide consisting essentially of a coagulationfactor and three to nine CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting essentially of a coagulation factor and three toten CTPs attached to the carboxy terminus of the coagulation factor.

In one embodiment, the present invention provides a polypeptideconsisting essentially of a coagulation factor and four gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting essentially of a coagulation factor and four tofive CTPs attached to the carboxy terminus of the coagulation factor. Inanother embodiment, provided herein is a polypeptide consistingessentially of a coagulation factor and four to six CTPs attached to thecarboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide consisting essentially of a coagulationfactor and four to seven CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting essentially of a coagulation factor and four toeight CTPs attached to the carboxy terminus of the coagulation factor.In another embodiment, provided herein is a polypeptide consistingessentially of a coagulation factor and four to nine CTPs attached tothe carboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide consisting essentially of a coagulationfactor and four to ten CTPs attached to the carboxy terminus of thecoagulation factor.

In one embodiment, the present invention provides a polypeptideconsisting essentially of a coagulation factor and five gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting essentially of a coagulation factor and five tosix CTPs attached to the carboxy terminus of the coagulation factor. Inanother embodiment, provided herein is a polypeptide consistingessentially of a coagulation factor and five to seven CTPs attached tothe carboxy terminus of the coagulation factor. In another embodiment,provided herein is a polypeptide consisting essentially of a coagulationfactor and five to eight CTPs attached to the carboxy terminus of thecoagulation factor. In another embodiment, provided herein is apolypeptide consisting essentially of a coagulation factor and five tonine CTPs attached to the carboxy terminus of the coagulation factor. Inanother embodiment, provided herein is a polypeptide consistingessentially of a coagulation factor and five to ten CTPs attached to thecarboxy terminus of the coagulation factor.

In another embodiment, provided herein is a polypeptide comprising,consisting essentially of, or consisting of a coagulation factor havingno CTPs on its amino terminus. In another embodiment, provided herein isa polypeptide comprising, consisting essentially of, or consisting of acoagulation factor lacking a CTP on its amino terminus. In anotherembodiment, provided herein is a polypeptide comprising, consistingessentially of, or consisting of a coagulation factor having at leastone CTP on its carboxy terminus and no CTPs on its amino terminus. Inanother embodiment, provided herein is a polypeptide comprising,consisting essentially of, or consisting of a coagulation factor havingthe number of CTPs on its carboxy terminus as described herein and noCTPs on its amino terminus.

In another embodiment, the present invention provides a polynucleotideencoding a polypeptide as described hereinabove.

In another embodiment, the present invention further provides acomposition comprising an expression vector comprising a polynucleotideencoding a CTP-modified polypeptide consisting of a Factor IX (FIX)polypeptide and three gonadotropin carboxy terminal peptides (CTPs)attached to the carboxy terminus of said FIX polypeptide.

In another embodiment, the present invention further provides apolynucleotide encoding a CTP-modified polypeptide consisting of aFactor VIIa (FVIIa) polypeptide and three gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of said FVIIapolypeptide.

In one embodiment, the present invention provides a recombinantcoagulation factor as described hereinabove. In one embodiment, thepresent invention provides an engineered coagulation factor as describedhereinabove. In one embodiment, the engineered coagulation factor asdescribed hereinabove is referred to as a CTP-modified coagulationfactor.

In one embodiment, the CTPs that are attached to the carboxy terminus ofthe coagulation factor are attached in tandem to the carboxy terminus.

In one embodiment, an engineered coagulation factor as described hereinhas equivalent or improved biological activity compared to thenon-CTP-modified coagulation factor. In another embodiment, anengineered coagulation factor as described herein has equivalent orimproved pharmacological measurements compared to the non-CTP-modifiedcoagulation factor. In another embodiment, an engineered coagulationfactor as described herein has equivalent or improved pharmacokineticscompared to the non-CTP-modified coagulation factor. In anotherembodiment, an engineered coagulation factor as described herein hasequivalent or improved pharmacodynamics compared to the non-CTP-modifiedcoagulation factor.

In one embodiment, the present invention provides a method of preventingor treating a clotting or coagulation disorder. In another embodiment,the present invention provides a method of preventing or treatinghemophilia in a subject comprising administering a CTP-modifiedcoagulation factor of the present invention. In another embodiment, thepresent invention provides a method of preventing and treatinghemophilia in a subject comprising administering a CTP-modifiedcoagulation factor of the present invention. In another embodiment, thepresent invention provides a method of treating hemophilia in a subjectcomprising administering a CTP-modified Factor VII of the presentinvention.

In another embodiment, the present invention provides a method oftreating hemophilia in a subject comprising administering a CTP-modifiedFactor IX of the present invention. In one embodiment, hemophilia ishemophilia B. In one embodiment, hemophilia B is known as factor IXdeficiency or Christmas disease. In one embodiment, the hemophilia issevere hemophilia, which in one embodiment, describes hemophilia inwhich the coagulation factor levels are 0-1%. In another embodiment, thehemophilia is moderate hemophilia, which in one embodiment, describeshemophilia in which the coagulation factor levels are 1-5%. In anotherembodiment, the hemophilia is mild hemophilia, which in one embodiment,describes hemophilia in which the coagulation factor levels are 5-50%.

In another embodiment, the present invention provides a method ofpreventing or treating a clotting or coagulation disorder in a subjectcomprising administering a CTP-modified Factor IX (FIX) polypeptidecomprising a FIX polypeptide and three chorionic gonadotropin carboxyterminal peptides (CTPs) attached to the carboxy terminus of said FIXpolypeptide to said subject, thereby preventing or treating a clottingor coagulation disorder in said subject. In another embodiment, thepresent invention provides a method of preventing or treating a clottingor coagulation disorder in a subject comprising administering aCTP-modified Factor VII (FVII) polypeptide comprising a FVII polypeptideand three chorionic gonadotropin carboxy terminal peptides (CTPs)attached to the carboxy terminus of said FVII polypeptide to saidsubject, thereby preventing or treating a clotting or coagulationdisorder in said subject.

In another embodiment, the present invention provides a method ofpreventing or treating hemophilia in a subject comprising administeringa CTP-modified Factor IX (FIX) polypeptide comprising a FIX polypeptideand three chorionic gonadotropin carboxy terminal peptides (CTPs)attached to the carboxy terminus of said FIX polypeptide to saidsubject, thereby preventing or treating hemophilia in said subject. Inanother embodiment, the present invention provides a method ofpreventing or treating hemophilia in a subject comprising administeringa CTP-modified Factor VIIa (FVIIa) polypeptide comprising a FVIIapolypeptide and three chorionic gonadotropin carboxy terminal peptides(CTPs) attached to the carboxy terminus of said FVIIa polypeptide tosaid subject, thereby preventing or treating hemophilia in said subject.

In another embodiment, the present invention provides a method oftreating hemophilia in a subject comprising administering one or moreCTP-modified coagulation factors as described herein to said subject.Thus, in one embodiment, the present invention provides a method oftreating hemophilia in a subject comprising administering a CTP-modifiedFactor IX (FIX) polypeptide comprising a FIX polypeptide and threechorionic gonadotropin carboxy terminal peptides (CTPs) attached to thecarboxy terminus of said FIX polypeptide and a CTP-modified Factor VIIa(FVIIa) polypeptide comprising a FVIIa polypeptide and three chorionicgonadotropin carboxy terminal peptides (CTPs) attached to the carboxyterminus of said FVIIa polypeptide to said subject, thereby treatinghemophilia in said subject. In one embodiment, the CTP-modified FIX andthe CTP-modified FVIIa are administered in the same composition at thesame time. In another embodiment, the CTP-modified FIX and theCTP-modified FVIIa are administered in separate compositions at the sametime. In another embodiment, the CTP-modified FIX and the CTP-modifiedFVIIa are administered in separate compositions at separate times.

In another embodiment, the present invention provides a method ofpreventing or treating hemophilia in a subject comprising administeringa CTP-modified Factor IX (FIX) or a CTP-modified Factor VII polypeptidecomprising a FIX or a FVII polypeptide and three chorionic gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FIX or said FVII polypeptide to said subject, thereby preventing ortreating hemophilia in said subject. In another embodiment, the presentinvention provides a method of preventing or treating hemophilia in asubject comprising administering a CTP-modified Factor IX (FIX) or aCTP-modified Factor VII polypeptide comprising a FIX or a FVIIpolypeptide and four chorionic gonadotropin carboxy terminal peptides(CTPs) attached to the carboxy terminus of said FIX or said FVIIpolypeptide to said subject, thereby preventing or treating hemophiliain said subject. In another embodiment, the present invention provides amethod of preventing or treating hemophilia in a subject comprisingadministering a CTP-modified Factor IX (FIX) or a CTP-modified FactorVII polypeptide comprising a FIX or a FVII polypeptide and fivechorionic gonadotropin carboxy terminal peptides (CTPs) attached to thecarboxy terminus of said FIX or said FVII polypeptide to said subject,thereby preventing or treating hemophilia in said subject. In anotherembodiment, the present invention provides a method of preventing ortreating hemophilia in a subject comprising administering a CTP-modifiedFactor IX (FIX) or a CTP-modified Factor VII polypeptide comprising aFIX or a FVII polypeptide and three to five chorionic gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FIX or said FVII polypeptide to said subject, thereby preventing ortreating hemophilia in said subject. In another embodiment, the presentinvention provides a method of preventing or treating hemophilia in asubject comprising administering a CTP-modified Factor IX (FIX) and aCTP-modified Factor VII polypeptide comprising a FIX and a FVIIpolypeptide and three chorionic gonadotropin carboxy terminal peptides(CTPs) attached to the carboxy terminus of said FIX and said FVIIpolypeptide to said subject, thereby preventing or treating hemophiliain said subject. In another embodiment, the present invention provides amethod of preventing or treating hemophilia in a subject comprisingadministering a CTP-modified Factor IX (FIX) and a CTP-modified FactorVII polypeptide comprising a FIX and a FVII polypeptide and three tofive chorionic gonadotropin carboxy terminal peptides (CTPs) attached tothe carboxy terminus of said FIX and said FVII polypeptide to saidsubject, thereby preventing or treating hemophilia in said subject.

In another embodiment, the present invention provides a method ofpreventing or treating hemophilia in a subject comprising subcutaneouslyor intravenously administering a CTP-modified Factor IX (FIX) or aCTP-modified Factor VII polypeptide comprising a FIX or a FVIIpolypeptide and three chorionic gonadotropin carboxy terminal peptides(CTPs) attached to the carboxy terminus of said FIX or said FVIIpolypeptide to said subject, thereby preventing or treating hemophiliain said subject. In another embodiment, the present invention provides amethod of preventing or treating hemophilia in a subject comprisingsubcutaneously or intravenously administering a CTP-modified Factor IX(FIX) or a CTP-modified Factor VII polypeptide comprising a FIX or aFVII polypeptide and four chorionic gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of said FIX or saidFVII polypeptide to said subject, thereby preventing or treatinghemophilia in said subject. In another embodiment, the present inventionprovides a method of preventing or treating hemophilia in a subjectcomprising subcutaneously or intravenously administering a CTP-modifiedFactor IX (FIX) or a CTP-modified Factor VII polypeptide comprising aFIX or a FVII polypeptide and five chorionic gonadotropin carboxyterminal peptides (CTPs) attached to the carboxy terminus of said FIX orsaid FVII polypeptide to said subject, thereby preventing or treatinghemophilia in said subject. In another embodiment, the present inventionprovides a method of preventing or treating hemophilia in a subjectcomprising subcutaneously or intravenously administering a CTP-modifiedFactor IX (FIX) or a CTP-modified Factor VII polypeptide comprising aFIX or a FVII polypeptide and three to five chorionic gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FIX or said FVII polypeptide to said subject, thereby preventing ortreating hemophilia in said subject. In another embodiment, the presentinvention provides a method of preventing or treating hemophilia in asubject comprising subcutaneously or intravenously administering aCTP-modified Factor IX (FIX) and a CTP-modified Factor VII polypeptidecomprising a FIX and a FVII polypeptide and three chorionic gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FIX and said FVII polypeptide to said subject, thereby preventingor treating hemophilia in said subject. In another embodiment, thepresent invention provides a method of preventing or treating hemophiliain a subject comprising subcutaneously or intravenously administering aCTP-modified Factor IX (FIX) and a CTP-modified Factor VII polypeptidecomprising a FIX and a FVII polypeptide and three to five chorionicgonadotropin carboxy terminal peptides (CTPs) attached to the carboxyterminus of said FIX and said FVII polypeptide to said subject, therebypreventing or treating hemophilia in said subject.

In some embodiments, provided herein is a method of preventing ortreating a hemophilia in a subject, the method comprising the step ofadministering to the subject a CTP-modified coagulation factor,comprising three to five chorionic gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of said coagulationfactor polypeptide, wherein the sequence of said CTP-modifiedcoagulation factor is selected from the group consisting of SEQ ID NO:25, 27, or 29, thereby preventing hemophilia in said subject.

In one embodiment, the present invention provides a cell comprising anexpression vector comprising a polynucleotide encoding a CTP-modifiedpolypeptide consisting of a Factor VII (FVII) polypeptide and three tofive gonadotropin carboxy terminal peptides (CTPs) attached to thecarboxy terminus of said FVII polypeptide. In another embodiment, thepresent invention provides a cell comprising an expression vectorcomprising a polynucleotide encoding a CTP-modified polypeptideconsisting of a Factor VII (FVII) polypeptide and three gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FVII polypeptide. In another embodiment, the present inventionprovides a cell comprising an expression vector comprising apolynucleotide encoding a CTP-modified polypeptide consisting of aFactor VII (FVII) polypeptide and five gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of said FVIIpolypeptide.

In one embodiment, the term “three to five” when referring togonadotropin carboxy terminal peptides (CTPs), refers to attachingthree, four, or five CTPs to the carboxy terminal of a coagulationfactor polypeptide provided herein

In one embodiment, the present invention provides a compositioncomprising an expression vector comprising a polynucleotide encoding aCTP-modified polypeptide consisting of a Factor VII (FVII) polypeptideand three to five gonadotropin carboxy terminal peptides (CTPs) attachedto the carboxy terminus of said FVII polypeptide. In another embodiment,the present invention provides a composition comprising an expressionvector comprising a polynucleotide encoding a CTP-modified polypeptideconsisting of a Factor VII (FVII) polypeptide and three gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FVII polypeptide. In another embodiment, the present inventionprovides a composition comprising an expression vector comprising apolynucleotide encoding a CTP-modified polypeptide consisting of aFactor VII (FVII) polypeptide and five gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of said FVIIpolypeptide.

In one embodiment, the present invention provides a method of extendingthe biological half-life of a Factor VII (FVII) polypeptide, comprisingthe step of attaching three to five chorionic gonadotropin carboxyterminal peptides (CTPs) to the carboxy terminus of said FVIIpolypeptide, thereby extending the biological half-life of said FVIIpolypeptide. In another embodiment, the present invention provides amethod of extending the biological half-life of a Factor VII (FVII)polypeptide, comprising the step of attaching three chorionicgonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus ofsaid FVII polypeptide, thereby extending the biological half-life ofsaid FVII polypeptide. In another embodiment, the present inventionprovides a method of extending the biological half-life of a Factor VII(FVII) polypeptide, comprising the step of attaching five chorionicgonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus ofsaid FVII polypeptide, thereby extending the biological half-life ofsaid FVII polypeptide.

In another embodiment, the present invention provides a method ofimproving the area under the curve (AUC) of a Factor VII (FVII)polypeptide, comprising the step of attaching three to five chorionicgonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus ofsaid FVII polypeptide, thereby improving the AUC of said FVIIpolypeptide. In another embodiment, the present invention provides amethod of improving the area under the curve (AUC) of a Factor VII(FVII) polypeptide, comprising the step of attaching three chorionicgonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus ofsaid FVII polypeptide, thereby improving the AUC of said FVIIpolypeptide. In another embodiment, the present invention provides amethod of improving the area under the curve (AUC) of a Factor VII(FVII) polypeptide, comprising the step of attaching five chorionicgonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus ofsaid FVII polypeptide, thereby improving the AUC of said FVIIpolypeptide.

In one embodiment, the present invention provides a method of reducingthe dosing frequency of a Factor VII (FVII) polypeptide, comprising thestep of attaching three to five chorionic gonadotropin carboxy terminalpeptides (CTPs) to the carboxy terminus of said FVII polypeptide,thereby reducing the dosing frequency of said FVII polypeptide. Inanother embodiment, the present invention provides a method of reducingthe dosing frequency of a Factor VII (FVII) polypeptide, comprising thestep of attaching three chorionic gonadotropin carboxy terminal peptides(CTPs) to the carboxy terminus of said FVII polypeptide, therebyreducing the dosing frequency of said FVII polypeptide. In anotherembodiment, the present invention provides a method of reducing thedosing frequency of a Factor VII (FVII) polypeptide, comprising the stepof attaching five chorionic gonadotropin carboxy terminal peptides(CTPs) to the carboxy terminus of said FVII polypeptide, therebyreducing the dosing frequency of said FVII polypeptide.

In one embodiment, the present invention provides a method of reducingthe clearance rate of a Factor VII (FVII) polypeptide, comprising thestep of attaching three to five chorionic gonadotropin carboxy terminalpeptides (CTPs) to the carboxy terminus of said FVII polypeptide,thereby reducing the clearance rate of said FVII polypeptide. In anotherembodiment, the present invention provides a method of reducing theclearance rate of a Factor VII (FVII) polypeptide, comprising the stepof attaching three chorionic gonadotropin carboxy terminal peptides(CTPs) to the carboxy terminus of said FVII polypeptide, therebyreducing the clearance rate of said FVII polypeptide. In anotherembodiment, the present invention provides a method of reducing theclearance rate of a Factor VII (FVII) polypeptide, comprising the stepof attaching five chorionic gonadotropin carboxy terminal peptides(CTPs) to the carboxy terminus of said FVII polypeptide, therebyreducing the clearance rate of said FVII polypeptide.

In one embodiment, the present invention provides a method of producinga CTP-modified Factor VII (FVII) polypeptide, comprising the step ofattaching three to five chorionic gonadotropin carboxy terminal peptides(CTPs) to the carboxy terminus of said FVII polypeptide, therebyproducing a CTP-modified FVII polypeptide. In another embodiment, thepresent invention provides a method of producing a CTP-modified FactorVII (FVII) polypeptide, comprising the step of attaching three chorionicgonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus ofsaid FVII polypeptide, thereby producing a CTP-modified FVIIpolypeptide. In another embodiment, the present invention provides amethod of producing a CTP-modified Factor VII (FVII) polypeptide,comprising the step of attaching five chorionic gonadotropin carboxyterminal peptides (CTPs) to the carboxy terminus of said FVIIpolypeptide, thereby producing a CTP-modified FVII polypeptide.

In another embodiment, the present invention provides a method oftreating hemophilia in a subject comprising administering a CTP-modifiedFactor VII (FVII) polypeptide comprising a FVII polypeptide and three tofive chorionic gonadotropin carboxy terminal peptides (CTPs) attached tothe carboxy terminus of said FVII polypeptide to said subject, therebytreating hemophilia in said subject. In another embodiment, the presentinvention provides a method of treating hemophilia in a subjectcomprising administering a CTP-modified Factor VII (FVII) polypeptidecomprising a FVII polypeptide and three chorionic gonadotropin carboxyterminal peptides (CTPs) attached to the carboxy terminus of said FVIIpolypeptide to said subject, thereby treating hemophilia in saidsubject. In another embodiment, the present invention provides a methodof treating hemophilia in a subject comprising administering aCTP-modified Factor VII (FVII) polypeptide comprising a FVII polypeptideand five chorionic gonadotropin carboxy terminal peptides (CTPs)attached to the carboxy terminus of said FVII polypeptide to saidsubject, thereby treating hemophilia in said subject.

In another embodiment, the methods provided herein further comprise thestep of attaching four chorionic gonadotropin carboxy terminal peptides(CTPs) to the carboxy terminus of said FVII polypeptide.

In one embodiment, the present invention provides a method of treatinghemophilia in a subject comprising administering a CTP-modifiedcoagulation factor of the present invention. In another embodiment, thepresent invention provides a method of treating hemophilia in a subjectcomprising administering a CTP-modified Factor IX of the presentinvention. In one embodiment, hemophilia is hemophilia B. In oneembodiment, hemophilia B is known as factor IX deficiency or Christmasdisease. In one embodiment, the hemophilia is severe hemophilia, whichin one embodiment, describes hemophilia in which the coagulation factorlevels are 0-1%. In another embodiment, the hemophilia is moderatehemophilia, which in one embodiment, describes hemophilia in which thecoagulation factor levels are 1-5%. In another embodiment, thehemophilia is mild hemophilia, which in one embodiment, describeshemophilia in which the coagulation factor levels are 5-50%.

In other embodiments, the engineered coagulation factor is for thetreatment of hemophilia B patients. In one embodiment, coagulationFactor IX comprising 3 CTPs in tandem in its carboxy terminus is for thetreatment of hemophilia B patients. In one embodiment, coagulationFactor IX comprising 4 CTPs in tandem in its carboxy terminus is for thetreatment of hemophilia B patients. In one embodiment, coagulationFactor IX comprising 5 CTPs in tandem in its carboxy terminus is for thetreatment of hemophilia B patients. In another embodiment, coagulationFactor IX comprising 2 CTPs in tandem in its carboxy terminus is for thetreatment of hemophilia B patients.

In another embodiment, coagulation Factor IX comprising 1 CTP repeat inits carboxy terminus is for the treatment of hemophilia B patients. Inother embodiments, the engineered coagulation factor can reduce thenumber of infusions required for a patient, reduce the required dosesfor a patient, or a combination thereof.

In one embodiment, coagulation Factor IX comprising 3 CTPs in tandem inits carboxy terminus exhibits an improved PK profile while maintainingits coagulation activity vs. FIX-CTP-CTP harvest, FIX-CTP harvest orrhFIX. In one embodiment, the elimination half-life of rFIX-CTP3 is 2.5-to 4-fold longer than rFIX in rats and in FIX-deficient mice. In oneembodiment, the administration of rFIX-CTP3 significantly prolonged theprocoagulatory effect in FIX-deficient mice for at least 76 hr afterdosing. In one embodiment, the administration of rFIX-CTP3 produced ahigher activity peak than rFIX in FIX-deficient mice. In anotherembodiment, coagulation Factor IX comprising 2 CTPs in tandem in itscarboxy terminus exhibits an improved PK profile while maintaining itscoagulation activity vs. FIX-CTP harvest or rhFIX. In anotherembodiment, coagulation Factor IX comprising 2 CTPs in tandem in itscarboxy terminus exhibits 3-fold increase in half-life and 4.5-foldhigher AUC compared to rhFIX.

In another embodiment, SC administration results in higherbioavailability of CTP-modified FVII as compared to recombinant FVII. Inanother embodiment, half-life is longer and bioavailability (AUC SC/AUCIV) is higher following FVIIa-CTP3 and 5 SC administration when comparedto SC administration of NovoSeven®. In another embodiment,subcutaneously injected MOD-5014 and MOD-5019 shows improved micesurvival in comparison to recombinant FYII (NovoSeven®) (see Example 8below).

In one embodiment, MOD-5014 is FVIIa-CTP₃ (having three CTP peptidesattached at the C-terminus end). In one embodiment, MOD-5014 provides along-acting coagulation factor. In one embodiment, MOD-5014 provides amore sustained and prolonged blood clotting response compared withrecombinant human FVIIa. (See for example, Example 14)

In one embodiment, deactivation of MOD-5014 by tissue factor pathwayinhibitor (TFPI) is dose-dependent. In one embodiment, deactivation ofMOD-5014 by TFPI shows a similar dose-dependent deactivation pattern tothat of recombinant FVIIa (NovoSeven®) by TFPI. In one embodiment,MOD-5014 is inhibitied by anti-thrombin III. In one embodiment,inhibition of MOD-5014 by anti-thrombin III is augmented in the presenceof heparin. In one embodiment, inhibition of MOD-5014 by anti-thrombinIII shows similar inhibition pattern to that of recombinant FVIIa(NovoSeven®), in the presence or absence of heparin. (see Example 11below)

In one embodiment, MOD-5014 generates thrombin in a dose-dependentmanner. In one embodiment, MOD-5014 decreases lag phase of thrombingeneration. In one embodiment, MOD-5014 decreases blood clotting tme. Inone embodiment, MOD-5014 increases efficiency of blood clot formation.In one embodiment, administration of MOD-5014 decreases blood clottingtime in a subject. In one embodiment, administration of MOD-5014increases efficiency of blood clot formation in a subject. In oneembodiment, the generation of thrombin by MOD-5014 is similar to thatproduced by recombinant FVIIa (NovoSeven®). In one embodiment, decreaseof lag-phase of thrombin generation by MOD-5014 is similar to thatproduced by recombinant FVIIa (NovoSeven®). In one embodiment, decreaseblood clotting time by MOD-5014 is similar to that produced byrecombinant FVIIa (NovoSeven®). In one embodiment, increased efficiencyof blood clot formation by MOD-5014 is similar to that produced byrecombinant FVIIa (NovoSeven®) (see Example 12 below).

As provided herein, CTP attachments to blood clotting factors, forexample factors FVII, FVIIA and FX, increase the half-life of the bloodclotting factor. Examples 11, 12 and 13 show that CTP attachments, forexample three CTPs attached to FVIIA, do not appear to affect bloodclotting activities. In one embodiment, CTP attachments to FVII do notinterfere with blood clot formation. In one embodiment, CTP attachmentsto FVII do not interfere with increased efficiency of blood clotformation. In one embodiment, CTP attachments to FVII do not interferewith decreased in blood clotting time. In one embodiment, binding ofphospholipid to FVII is maintained following attachment of CTPs to theblood clotting factor. In one embodiment, CTP attachments to FVIIA donot interfere with blood clot formation. In one embodiment, CTPattachments to FVIIA do not interfere with increased efficiency of bloodclot formation. In one embodiment, CTP attachments to FVIIA do notinterfere with decreased blood clot formation. In one embodiment,binding of phospholipid to FVIIA is maintained following attachment ofCTPs to the blood clotting factor.

In another embodiment, the present invention provides a method oftreating hemophilia in a subject comprising administering a CTP-modifiedFactor IX (FIX) polypeptide comprising a FIX polypeptide and three tofive chorionic gonadotropin carboxy terminal peptides (CTPs) attached tothe carboxy terminus of said FIX polypeptide to said subject, therebytreating hemophilia in said subject. In another embodiment, the presentinvention provides a method of treating hemophilia in a subjectcomprising administering a CTP-modified Factor IX (FIX) polypeptidecomprising a FIX polypeptide and three chorionic gonadotropin carboxyterminal peptides (CTPs) attached to the carboxy terminus of said FIXpolypeptide to said subject, thereby treating hemophilia in saidsubject. In another embodiment, the present invention provides a methodof treating hemophilia in a subject comprising administering aCTP-modified Factor IX (FIX) polypeptide comprising a FIX polypeptideand five chorionic gonadotropin carboxy terminal peptides (CTPs)attached to the carboxy terminus of said FIX polypeptide to saidsubject, thereby treating hemophilia in said subject. In anotherembodiment, the present invention provides a method of treatinghemophilia in a subject comprising administering a CTP-modified FactorVIIa (FVIIa) polypeptide comprising a FVIIa polypeptide and three tofive chorionic gonadotropin carboxy terminal peptides (CTPs) attached tothe carboxy terminus of said FVIIa polypeptide to said subject, therebytreating hemophilia in said subject.

In another embodiment, the present invention provides a method oftreating hemophilia in a subject comprising administering one or moreCTP-modified coagulation factors as described herein to said subject.Thus, in one embodiment, the present invention provides a method oftreating hemophilia in a subject comprising administering a CTP-modifiedFactor IX (FIX) polypeptide comprising a FIX polypeptide and threechorionic gonadotropin carboxy terminal peptides (CTPs) attached to thecarboxy terminus of said FIX polypeptide and a CTP-modified Factor VIIa(FVIIa) polypeptide comprising a FVIIa polypeptide and three to fivechorionic gonadotropin carboxy terminal peptides (CTPs) attached to thecarboxy terminus of said FVIIa polypeptide to said subject, therebytreating hemophilia in said subject. In one embodiment, the CTP-modifiedFIX and the CTP-modified FVIIa are administered in the same compositionat the same time. In another embodiment, the CTP-modified FIX and theCTP-modified FVIIa are administered in separate compositions at the sametime. In another embodiment, the CTP-modified FIX and the CTP-modifiedFVIIa are administered in separate compositions at separate times.

In other embodiments, the engineered coagulation factor is for thetreatment of hemophilia B patients. In one embodiment, coagulationFactor IX comprising 3 CTPs in tandem in its carboxy terminus is for thetreatment of hemophilia B patients. In one embodiment, coagulationFactor IX comprising 4 CTPs in tandem in its carboxy terminus is for thetreatment of hemophilia B patients. In one embodiment, coagulationFactor IX comprising 5 CTPs in tandem in its carboxy terminus is for thetreatment of hemophilia B patients. In another embodiment, coagulationFactor IX comprising 2 CTPs in tandem in its carboxy terminus is for thetreatment of hemophilia B patients. In another embodiment, coagulationFactor IX comprising 1 CTP repeat in its carboxy terminus is for thetreatment of hemophilia B patients. In other embodiments, the engineeredcoagulation factor can reduce the number of infusions required for apatient, reduce the required doses for a patient, or a combinationthereof.

Example 14 shows the results of administering MOD-5014 to a large mammal(dogs). MOD-5014 administration provided an effective and safelong-acting FVIIa for blood coagulation. Treatment using MOD-5014 may beprophylactic or on-demand. In one embodiment, the present inventionprovides a method of treating hemophilia in a subject comprisingadministering MOD-5014 to said subject, thereby treating hemophilia insaid subject. In one embodiment, the present invention provides a methodof preventing excess bleeding in a subject comprising administeringMOD-5014 to said subject, thereby preventing excess bleeding in saidsubject. In one embodiment, the present invention provides a method ofprophylacticly treating hemophilia in a subject comprising administeringMOD-5014 to said subject, thereby prophylactically treating hemophiliain said subject.

In one embodiment, treating hemophilia in a subject with MOD-5014comprises a reduced frequency of administration of MOD-5014, as comparedwith recombinant FVIIa (NovoSeven®). In one embodiment, prophylacticallytreating hemophilia in a subject with MOD-5014 comprises a reducedfrequency of administration of MOD-5014, as compared with recombinantFVIIa (NovoSeven®). In one embodiment, preventing excess bleeding in asubject with MOD-5014 comprises a reduced frequency of administration ofMOD-5014, as compared with recombinant FVIIa (NovoSeven®).

In one embodiment, coagulation Factor IX comprising 3 CTPs in tandem inits carboxy terminus exhibits an improved PK profile while maintainingits coagulation activity vs. FIX-CTP-CTP harvest, FIX-CTP harvest orrhFIX. In one embodiment, the elimination half-life of rFIX-CTP3 is 2.5-to 4-fold longer than rFIX in rats and in FIX-deficient mice. In oneembodiment, the administration of rFIX-CTP3 significantly prolonged theprocoagulatory effect in FIX-deficient mice for at least 76 hr afterdosing. In one embodiment, the administration of rFIX-CTP3 produced ahigher activity peak than rFIX in FIX-deficient mice. In anotherembodiment, coagulation Factor IX comprising 2 CTPs in tandem in itscarboxy terminus exhibits an improved PK profile while maintaining itscoagulation activity vs. FIX-CTP harvest or rhFIX. In anotherembodiment, coagulation Factor IX comprising 2 CTPs in tandem in itscarboxy terminus exhibits 3-fold increase in half-life and 4.5-foldhigher AUC compared to rhFIX.

In one embodiment, coagulation Factor VII comprising 3 CTPs in tandem inits carboxy terminus exhibits an improved PK profile while maintainingits coagulation activity vs. NovoSeven® (see Table 59 and FIG. 36).

In another embodiment, the terms “CTP peptide,” “carboxy terminalpeptide” and “CTP sequence” are used interchangeably herein. In anotherembodiment, the carboxy terminal peptide is a full-length CTP. Eachpossibility represents a separate embodiment of the invention.

In another embodiment, a signal peptide is attached to the aminoterminus of the CTP, as described in U.S. Pat. No. 7,553,940, which isincorporated by reference herein in its entirety.

In other embodiments, the term engineered coagulation factor refers tothe amino acid sequence of a matured coagulation factor. In otherembodiments, the term engineered coagulation factor refers to the aminoacid sequence of the coagulation factor including its signal sequence orsignal peptide.

In another embodiment, “signal sequence” and “signal peptide” are usedinterchangeably herein. In another embodiment, “sequence” when inreference to a polynucleotide molecule can refer to a coding portion.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, an engineered coagulation factor comprising atleast one CTP as described herein has enhanced in vivo biologicalactivity compared the same coagulation factor without at least one CTP.In one embodiment, the enhanced biological activity stems from thelonger half-life of the engineered coagulation factor while maintainingat least some biological activity. In another embodiment, the enhancedbiological activity stems from enhanced biological activity resultingfrom the CTP modification. In another embodiment, the enhancedbiological activity stems from both a longer half-life and from enhancedfunctionality of the CTP-modified coagulation factor.

In some embodiments, at least one CTP sequence at the carboxy terminalend of the coagulation factor provides enhanced protection againstdegradation of a coagulation factor. In some embodiments, at least oneCTP sequence at the carboxy terminal end of the coagulation factorprovides enhanced protection against clearance. In some embodiments, atleast one CTP sequence at the carboxy terminal end of the coagulationfactor provides prolonged clearance time. In some embodiments, at leastone CTP sequence at the carboxy terminal end of the coagulation factorenhances its Cmax. In some embodiments, at least one CTP sequence at thecarboxy terminal end of the coagulation factor enhances its Tmax. Insome embodiments, at least one CTP sequence at the carboxy terminal endof the coagulation factor prolongs its T½.

In another embodiment, a conjugated coagulation factor of this inventionis used in the same manner as an unmodified conjugated coagulationfactor. In another embodiment, a conjugated coagulation factor of thisinvention has an increased circulating half-life and plasma residencetime, decreased clearance, and increased clinical activity in vivo. Inanother embodiment, due to the improved properties of the conjugatedcoagulation factor as described herein, this conjugate is administeredless frequently than the unmodified form of the same coagulation factor.

In another embodiment, decreased frequency of administration will resultin improved treatment strategy, which in one embodiment, will lead toimproved patient compliance leading to improved treatment outcomes, aswell as improved patient quality of life. In another embodiment,compared to conventional conjugates of coagulation factors, it has beenfound that conjugates having the molecular weight and linker structureof the conjugates of this invention have an improved potency, improvedstability, elevated AUC levels, and enhanced circulating half-life.

In another embodiment, the present invention further provides apharmaceutical composition comprising a CTP-modified Factor IX (FIX)polypeptide consisting of a FIX polypeptide and three gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid CTP-modified FIX polypeptide.

In another embodiment, the present invention further provides apharmaceutical composition comprising a CTP-modified Factor VIIa (FVIIa)polypeptide consisting of a FVIIa polypeptide and three gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FVIIa.

In another embodiment, the present invention further provides apharmaceutical composition comprising a CTP-modified Factor VIIa (FVIIa)polypeptide consisting of a FVIIa polypeptide and four gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FVIIa.

In another embodiment, the present invention further provides apharmaceutical composition comprising a CTP-modified Factor VIIa (FVIIa)polypeptide consisting of a FVIIa polypeptide and five gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FVIIa.

In another embodiment, provided herein is a composition comprising aconjugated coagulation factor as described herein. In anotherembodiment, provided herein is a pharmaceutical composition comprisingthe conjugated coagulation factor as described herein. In anotherembodiment, provided herein is a pharmaceutical composition comprising atherapeutically effective amount of the conjugated coagulation factor asdescribed herein. In one embodiment, a therapeutically effective amountof a conjugated coagulation factor is determined according to factorssuch as the specific condition being treated, the condition of thepatient being treated, as well as the other ingredients in thecomposition.

In another embodiment, a conjugated coagulation factor as describedherein is useful in the treatment of subjects afflicted with acoagulation or clotting disorder. In another embodiment, the coagulationor clotting disorder is Hemophilia. In another embodiment, a conjugatedcoagulation factor as described herein is useful in the prophylactictherapy of Hemophilia thus reducing the risk of bleeding and associatedcomplications. In another embodiment, a conjugated coagulation factor asdescribed herein is useful in the treatment of subjects afflicted withHemophilia while reducing the risk of developing inhibitory antibodiesto exogenously administered coagulation factors. In another embodiment,a conjugated coagulation factor as described herein is useful in thetreatment of subjects afflicted with Hemophilia thus inducinghomeostasis.

In one embodiment, a CTP-modified coagulation factor of the presentinvention has therapeutic uses. In another embodiment, a CTP-modifiedcoagulation factor of the present invention has prophylactic uses.

In another embodiment, a conjugated coagulation factor as describedherein is useful in the treatment of subjects experiencing excessivebleeding or bruising or having a prolonged Prothrombin Time (PT) orPartial Thromboplastin Time (PTT). In another embodiment, a conjugatedcoagulation factor as described herein is useful in the treatment ofsubjects having an acquired condition that is causing bleeding, such asvitamin K deficiency or liver disease. In another embodiment, aconjugated coagulation factor as described herein is useful in thetreatment of subjects having deficiencies in coagulation factors thatare acquired (due to other diseases) or inherited, mild or severe,permanent or temporary. In another embodiment, a conjugated coagulationfactor as described herein is useful in the treatment of subjectsafflicted with hemophilia A. In another embodiment, a conjugatedcoagulation factor as described herein is useful in the treatment ofsubjects afflicted with hemophilia B. In another embodiment, aconjugated coagulation factor as described herein is useful in thetreatment of subjects having acquired deficiencies due to chronicdiseases, such as liver disease or cancer; to an acute condition such asdisseminated intravascular coagulation (DIC), which uses up clottingfactors at a rapid rate; or to a deficiency in vitamin K or treatmentwith a vitamin K antagonist like warfarin (the production of factors II,VII, IX, and X require vitamin K). In another embodiment, a conjugatedcoagulation factor as described herein is useful in the treatment ofsubjects afflicted with a disease in which causes clotting imbalancessuch as but not limited to: a liver disease, uremia, a cancer, a bonemarrow disorder, an exposure to snake venom, a vitamin K deficiency, ananticoagulation therapy, an accidental ingestion of the anticoagulantwarfarin, multiple blood transfusions (stored units of blood lose someof their clotting factors), or a combination thereof. In anotherembodiment, the present invention provides a method of treating deepvein thrombosis in a subject comprising administering a CTP-modifiedcoagulation factor of the present invention. In another embodiment, thepresent invention provides a method of preventing uncontrolled bleedingin a subject with hemophilia comprising administering a CTP-modifiedcoagulation factor of the present invention. In another embodiment, thepresent invention provides a method of preventing bleeding episodes in asubject with hemophilia comprising administering a CTP-modifiedcoagulation factor of the present invention. In another embodiment, thepresent invention provides a method of controlling bleeding episodes ina subject with hemophilia B (congenital factor IX deficiency).

In another embodiment, the compositions and methods of the presentinvention are for the treatment of bleeding episodes in hemophilia A orB patients with inhibitors to FVIII or FIX and in patients with acquiredhemophilia; prevention of bleeding in surgical interventions or invasiveprocedures in hemophilia A or B patients with inhibitors to FVIII or FIXand in patients with acquired hemophilia; treatment of bleeding episodesin patients with congenital FVII deficiency and prevention of bleedingin surgical interventions or invasive procedures in patients withcongenital FVII deficiency. Acquired hemophilia is a spontaneousautoimmune disorder in which patients with previously normal hemostasisdevelop autoantibodies against clotting factors, most frequently FVIII.The development of autoantibodies against FVIII leads to FVIIIdeficiency, which results in insufficient generation of thrombin byfactor IXa and the factor VIIIa complex through the intrinsic pathway ofthe coagulation cascade. The following conditions may be associated withacquired hemophilia A: idiopathic, pregnancy, autoimmune disorders,inflammatory bowel disease, ulcerative colitis, dermatologic disorders(eg, psoriasis, pemphigus), respiratory diseases (eg, asthma, chronicobstructive pulmonary disease), allergic drug reactions, diabetes, acutehepatitis B infection, acute hepatitis C infection, malignancies-solidtumors (prostate, lung, colon, pancreas, stomach, bile duct, head andneck, cervix, breast, melanoma, kidney), hematologic malignancies. Itwill be appreciated by the skilled artisan that autoimmune disorders mayinclude rheumatoid arthritis, systemic lupus erythematosus, multiplesclerosis, temporal arteritis, sjögren syndrome, autoimmune hemolyticanemia, goodpasture syndrome, myasthenia gravis, graves' disease,autoimmune hypothyroidism. It will be appreciated by the skilled artisanthat allergic reactions may occur from a subject being administeredpenicillin and its derivatives, sulfamides, phenyloin, chloramphenicol,methyldopa, depot thioxanthene, interferon alfa, fludarabine, bacillecalmette-guérin (BCG) vaccination, desvenlafaxine. It will beappreciated by the skilled artisan that hematologic malignancies mayinclude chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiplemyeloma, waldenstrom macroglobulinemia, myelodysplastic syndrome,myelofibrosis, and erythroleukemia. Hence, and in one embodiment,provided herein is a method for treating acquired hemophilia in asubject, comprising administering to the subject any of the compositionsprovided herein.

In another embodiment, the compositions and methods of the presentinvention are for the treatment or prevention of muscle bleeds. Inanother embodiment, the compositions and methods of the presentinvention are for the treatment or prevention of joint bleeds. Inanother embodiment, the compositions and methods of the presentinvention provide therapeutic or prophylactic treatment of epistaxis andgum bleeding, mucous membrane bleeding, bleeding into the centralnervous system. In another embodiment, the compositions and methods ofthe present invention provide therapeutic or prophylactic treatment ofgastrointestinal or cerebral bleeding. In another embodiment, thecompositions and methods of the present invention provide therapeutic orprophylactic treatment of low frequency mild bleeds. In anotherembodiment, the compositions and methods of the present inventionprovide therapeutic or prophylactic treatment of low frequency moderatebleeds. In another embodiment, the compositions and methods of thepresent invention provide therapeutic or prophylactic treatment of highfrequency mild bleeds. In another embodiment, the compositions andmethods of the present invention provide therapeutic or prophylactictreatment of high frequency moderate bleeds.

In one embodiment, the compositions and methods of the present inventionprovide therapeutic or prophylactic treatment of asymptomatichemophilia. In another embodiment, the compositions and methods of thepresent invention provide therapeutic or prophylactic treatment of mildto moderate hemophilia. In another embodiment, the compositions andmethods of the present invention provide therapeutic or prophylactictreatment of severe hemophilia.

In one embodiment, the compositions and methods of the present inventionprovide therapeutic or prophylactic treatment of hemorrhage, which inone embodiment, is uncontrollable hemorrhage, and, in anotherembodiment, intracerebral hemorrhage. In another embodiment, thecompositions and methods of the present invention provide therapeutic orprophylactic treatment of neonatal coagulopathies; severe hepaticdisease; high-risk surgical procedures; traumatic blood loss; bonemarrow transplantation; thrombocytopenias and platelet functiondisorders; urgent reversal of oral anticoagulation; congenitaldeficiencies of factors V, VII, X, and XI; or von Willebrand disease, inone embodiment, von Willebrand disease with inhibitors to von Willebrandfactor.

In one embodiment, a CTP-modified coagulation factor of the presentinvention is for the treatment of hemophilia or a related disease asdescribed herein in a subject. In one embodiment, the subject is human.In another embodiment, the subject is a human child. In anotherembodiment, the subject is a domesticated animal. In another embodiment,the subject is a mammal. In another embodiment, the subject is a farmanimal. In another embodiment, the subject is a monkey. In anotherembodiment, the subject is a horse. In another embodiment, the subjectis a cow. In another embodiment, the subject is a mouse. In anotherembodiment, the subject is a rat. In another embodiment, the subject iscanine. In another embodiment, the subject is feline. In anotherembodiment, the subject is bovine, ovine, porcine, equine, murine, orcervine. In one embodiment, the subject is male. In another embodiment,the subject is female. In one embodiment, the subject is a child, inanother embodiment, an adolescent, in another embodiment, an adult or,in another embodiment, an elderly subject. In another embodiment, thesubject is a pediatric subject, in another embodiment, a geriatricsubject.

In another embodiment, a [(CTP)n>1-coagulation factor] as describedherein comprises a full length coagulation factor or an active fragmentthereof connected via a peptide bond on its carboxy terminus to at leastone CTP unit with no CTPs on its amino terminus. In another embodiment,a [(CTP)n>1-coagulation factor] as described herein comprises acoagulation factor or an active fragment thereof connected via a peptidebond to at least one CTP unit which is connected to an additional CTPunit via a peptide bond with no CTPs on its amino terminus. In anotherembodiment, one nucleic acid molecule encodes an engineered coagulationfactor comprising at least one CTP attached to its C-terminus and noCTPs on its amino terminus.

In another embodiment, the CTP is attached to the coagulation factor viaa linker. In another embodiment, the linker which connects the CTPsequence to the coagulation factor is a covalent bond. In anotherembodiment, the linker which connects the CTP sequence to thecoagulation factor is a peptide bond. In another embodiment, the linkerwhich connects the CTP sequence to the coagulation factor is asubstituted peptide bond. In another embodiment, the CTP sequencecomprises: DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 1). In anotherembodiment, the CTP sequence comprises: SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQ ID NO: 2). In another embodiment, the CTP sequence comprises anamino acid sequence selected from the sequences set forth in SEQ ID NO:1 and SEQ ID NO: 2.

In another embodiment, the carboxy terminal peptide (CTP) peptide of thepresent invention comprises the amino acid sequence from amino acid 112to position 145 of human chorionic gonadotropin, as set forth in SEQ IDNO: 1. In another embodiment, the CTP sequence of the present inventioncomprises the amino acid sequence from amino acid 118 to position 145 ofhuman chorionic gonadotropin, as set forth in SEQ ID NO: 2. In anotherembodiment, the CTP sequence also commences from any position betweenpositions 112-118 and terminates at position 145 of human chorionicgonadotropin. In some embodiments, the CTP sequence peptide is 28, 29,30, 31, 32, 33 or 34 amino acids long and commences at position 112,113, 114, 115, 116, 117 or 118 of the CTP amino acid sequence.

In another embodiment, the CTP peptide is a variant of chorionicgonadotropin CTP which differs from the native CTP by 1-5 conservativeamino acid substitutions as described in U.S. Pat. No. 5,712,122, whichis incorporated herein by reference. In another embodiment, the CTPpeptide is a variant of chorionic gonadotropin CTP which differs fromthe native CTP by 1 conservative amino acid substitution. In anotherembodiment, the CTP peptide is a variant of chorionic gonadotropin CTPwhich differs from the native CTP by 2 conservative amino acidsubstitutions. In another embodiment, the CTP peptide is a variant ofchorionic gonadotropin CTP which differs from the native CTP by 3conservative amino acid substitutions. In another embodiment, the CTPpeptide is a variant of chorionic gonadotropin CTP which differs fromthe native CTP by 4 conservative amino acid substitutions. In anotherembodiment, the CTP peptide is a variant of chorionic gonadotropin CTPwhich differs from the native CTP by 5 conservative amino acidsubstitutions.

In another embodiment, the CTP peptide amino acid sequence of thepresent invention is at least 70% homologous to the native CTP aminoacid sequence or a peptide thereof. In another embodiment, the CTPpeptide amino acid sequence of the present invention is at least 80%homologous to the native CTP amino acid sequence or a peptide thereof.In another embodiment, the CTP peptide amino acid sequence of thepresent invention is at least 90% homologous to the native CTP aminoacid sequence or a peptide thereof. In another embodiment, the CTPpeptide amino acid sequence of the present invention is at least 95%homologous to the native CTP amino acid sequence or a peptide thereof.In another embodiment, the CTP peptide amino acid sequence of thepresent invention is at least 98% homologous to the native CTP aminoacid sequence or a peptide thereof.

In another embodiment, the polynucleotide encoding the CTP peptide ofthe present invention is at least 70% homologous to the native human CTPDNA sequence or a peptide thereof. In another embodiment, thepolynucleotide encoding the CTP peptide of the present invention is atleast 80% homologous to the native human CTP DNA sequence or a peptidethereof. In another embodiment, the polynucleotide encoding the CTPpeptide of the present invention is at least 90% homologous to thenative CTP DNA sequence or a peptide thereof. In another embodiment, thepolynucleotide encoding the CTP peptide of the present invention is atleast 95% homologous to the native CTP DNA sequence or a peptidethereof. In another embodiment, the polynucleotide encoding the CTPpeptide of the present invention is at least 98% homologous to thenative CTP DNA sequence or a peptide thereof.

In one embodiment, at least one of the chorionic gonadotropin CTP aminoacid sequences is truncated. In another embodiment, both of thechorionic gonadotropin CTP amino acid sequences are truncated. Inanother embodiment, 2 of the chorionic gonadotropin CTP amino acidsequences are truncated. In another embodiment, 3 of the chorionicgonadotropin CTP amino acid sequences are truncated. In anotherembodiment, 4 of the chorionic gonadotropin CTP amino acid sequences aretruncated. In another embodiment, 5 of the chorionic gonadotropin CTPamino acid sequences are truncated. In another embodiment, 2 or more ofthe chorionic gonadotropin CTP amino acid sequences are truncated. Inanother embodiment, all of the chorionic gonadotropin CTP amino acidsequences are truncated. In one embodiment, the truncated CTP comprisesthe first 10 amino acids of SEQ ID NO: 3. In another embodiment, SEQ IDNO: 3 comprises the following amino acid (AA) sequence: SSSSKAPPPSLP.

In one embodiment, the truncated CTP comprises the first 10 amino acidsof SEQ ID NO: 4. In another embodiment, SEQ ID NO: 4 comprises thefollowing amino acid (AA) sequence: SSSSKAPPPSLPSPSRLPGPSDTPILPQ.

In one embodiment, the truncated CTP comprises the first 11 amino acidsof SEQ ID NO: 4. In one embodiment, the truncated CTP comprises thefirst 12 amino acids of SEQ ID NO: 4. In one embodiment, the truncatedCTP comprises the first 8 amino acids of SEQ ID NO: 4 or SEQ ID NO: 3.In one embodiment, the truncated CTP comprises the first 13 amino acidsof SEQ ID NO: 4. In one embodiment, the truncated CTP comprises thefirst 14 amino acids of SEQ ID NO: 4. In one embodiment, the truncatedCTP comprises the first 6 amino acids of SEQ ID NO: 4 or SEQ ID NO: 3.In one embodiment, the truncated CTP comprises the first 5 amino acidsof SEQ ID NO: 4 or SEQ ID NO: 3.

In one embodiment, at least one of the chorionic gonadotropin CTP aminoacid sequences is glycosylated. In another embodiment, both of thechorionic gonadotropin CTP amino acid sequences are glycosylated. Inanother embodiment, 2 of the chorionic gonadotropin CTP amino acidsequences are glycosylated. In another embodiment, 3 of the chorionicgonadotropin CTP amino acid sequences are glycosylated. In anotherembodiment, 4 of the chorionic gonadotropin CTP amino acid sequences areglycosylated. In another embodiment, 5 of the chorionic gonadotropin CTPamino acid sequences are glycosylated. In another embodiment, 2 or moreof the chorionic gonadotropin CTP amino acid sequences are glycosylated.In another embodiment, all of the chorionic gonadotropin CTP amino acidsequences are glycosylated.

In one embodiment, the CTP sequence of the present invention comprisesat least one glycosylation site. In one embodiment, the CTP sequence ofthe present invention comprises 2 glycosylation sites. In oneembodiment, the CTP sequence of the present invention comprises 3glycosylation sites. In one embodiment, the CTP sequence of the presentinvention comprises 4 glycosylation sites. In one embodiment, one ormore of the chorionic gonadotropin CTP amino acid sequences is fullyglycosylated. In another embodiment, one or more of the chorionicgonadotropin CTP amino acid sequences is partially glycosylated. In oneembodiment, partially glycosylated indicates that one of the CTPglycosylation sites is glycosylated. In another embodiment, two of theCTP glycosylation sites are glycosylated. In another embodiment, threeof the CTP glycosylation sites are glycosylated.

In some embodiments, the CTP sequence modification is advantageous inpermitting the usage of lower dosages. In some embodiments, the CTPsequences modification is advantageous in permitting fewer dosages. Insome embodiments, the CTP sequences modification is advantageous inpermitting a safe, long-acting effect.

In some embodiments, “polypeptide”, “engineered coagulation factor”, or“protein” as used herein encompasses native polypeptides (eitherdegradation products, synthetically synthesized polypeptides orrecombinant polypeptides) and peptidomimetics (typically, syntheticallysynthesized polypeptides), as well as peptoids and semipeptoids whichare polypeptide analogs, which have, in some embodiments, modificationsrendering the polypeptides comprising a coagulation factor even morestable while in a body or more capable of penetrating into cells.

In some embodiments, modifications include, but are limited to Cterminus modification, polypeptide bond modification, including, but notlimited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O, CH2-CH2, S═C—NH,CH═CH or CF═CH, backbone modifications, and residue modification.Methods for preparing peptidomimetic compounds are well known in the artand are specified, for example, in Quantitative Drug Design, C. A.Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which isincorporated by reference as if fully set forth herein. Further detailsin this respect are provided hereinunder.

In some embodiments, polypeptide bonds (—CO—NH—) within the polypeptideare substituted. In some embodiments, the polypeptide bonds aresubstituted by N-methylated bonds (—N(CH3)-CO—). In some embodiments,the polypeptide bonds are substituted by ester bonds(—C(R)H—C—O—C(R)—N—). In some embodiments, the polypeptide bonds aresubstituted by ketomethylen bonds (—CO—CH2-). In some embodiments, thepolypeptide bonds are substituted by α-aza bonds (—NH—N(R)—CO—), whereinR is any alkyl, e.g., methyl, carba bonds (—CH2-NH—). In someembodiments, the polypeptide bonds are substituted by hydroxyethylenebonds (—CH(OH)—CH2-). In some embodiments, the polypeptide bonds aresubstituted by thioamide bonds (—CS—NH—). In some embodiments, thepolypeptide bonds are substituted by olefinic double bonds (—CH═CH—). Insome embodiments, the polypeptide bonds are substituted by retro amidebonds (—NH—CO—). In some embodiments, the polypeptide bonds aresubstituted by polypeptide derivatives (—N(R)—CH2-CO—), wherein R is the“normal” side chain, naturally presented on the carbon atom. In someembodiments, these modifications occur at any of the bonds along thepolypeptide chain and in one embodiment at several (2-3 bonds) at thesame time.

In some embodiments, natural aromatic amino acids of the polypeptidesuch as Trp, Tyr and Phe, are substituted for synthetic non-natural acidsuch as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylatedderivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. Insome embodiments, the polypeptides of the present invention include oneor more modified amino acid or one or more non-amino acid monomers (e.g.fatty acid, complex carbohydrates etc).

In one embodiment, “amino acid” or “amino acid sequence” is understoodto include the 20 naturally occurring amino acid; those amino acid oftenmodified post-translationally in vivo, including, for example,hydroxyproline, phosphoserine and phosphothreonine; and other unusualamino acid including, but not limited to, 2-aminoadipic acid,hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Inone embodiment, “amino acid” includes both D- and L-amino acids.

In some embodiments, the polypeptides of the present invention areutilized in therapeutics which requires the polypeptides comprising acoagulation factor to be in a soluble form. In some embodiments, thepolypeptides of the present invention include one or more non-natural ornatural polar amino acid, including but not limited to serine andthreonine which are capable of increasing polypeptide solubility due totheir hydroxyl-containing side chain.

In some embodiments, the engineered coagulation factor of the presentinvention is utilized in a linear form, although it will be appreciatedby one skilled in the art that in cases where cyclicization does notseverely interfere with engineered coagulation factors characteristics,cyclic forms of the engineered coagulation factors can also be utilized.

In some embodiments, the engineered coagulation factors of the presentinvention are biochemically synthesized such as by using standard solidphase techniques. In some embodiments, these biochemical methods includeexclusive solid phase synthesis, partial solid phase synthesis, fragmentcondensation, or classical solution synthesis.

In some embodiments, recombinant protein techniques are used to generatethe engineered coagulation factors of the present invention. In someembodiments, recombinant protein techniques are used for the generationof relatively long polypeptides (e.g., longer than 18-25 amino acids).In some embodiments, recombinant protein techniques are used for thegeneration of large amounts of the engineered coagulation factors of thepresent invention. In some embodiments, recombinant techniques aredescribed by Bitter et al., (1987) Methods in Enzymol. 153:516-544,Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al.(1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311,Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984)Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 andWeissbach & Weissbach, 1988, Methods for Plant Molecular Biology,Academic Press, NY, Section VIII, pp 421-463, which are incorporatedherein by reference in their entirety.

In another embodiment, the invention provides a polynucleotide moleculecomprising the coding portion of a gene encoding a polypeptidecomprising a coagulation factor and gonadotropin carboxy terminalpeptides attached to the carboxy terminus of the coagulation factor, asdescribed hereinabove. In another embodiment, the invention provides apolynucleotide molecule consisting of the coding portion of a geneencoding a polypeptide comprising a coagulation factor and gonadotropincarboxy terminal peptides attached to the carboxy terminus of thecoagulation factor, as described hereinabove. In another embodiment, theinvention provides a polynucleotide molecule consisting essentially ofthe coding portion of a gene encoding a polypeptide comprising acoagulation factor and gonadotropin carboxy terminal peptides attachedto the carboxy terminus of the coagulation factor, as describedhereinabove.

In another embodiment, the invention provides a polynucleotide encodinga polypeptide comprising a coagulation factor and three gonadotropincarboxy terminal peptides attached to the carboxy terminus of thecoagulation factor, as described hereinabove. In another embodiment, theinvention provides a polynucleotide encoding a polypeptide consisting ofa coagulation factor and three gonadotropin carboxy terminal peptidesattached to the carboxy terminus of the coagulation factor, as describedhereinabove. In another embodiment, the invention provides apolynucleotide encoding a polypeptide consisting essentially of acoagulation factor and three gonadotropin carboxy terminal peptidesattached to the carboxy terminus of the coagulation factor, as describedhereinabove. In one embodiment, the polynucleotide is a polynucleotidesequence. In one embodiment, the polynucleotide is a polynucleotidemolecule.

In another embodiment, the invention provides an expression vectorcomprising a polynucleotide molecule as described herein. In anotherembodiment, the present invention provides an expression vectorcomprising a polynucleotide encoding a CTP-modified polypeptideconsisting of a Factor IX (FIX) polypeptide and three gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FIX polypeptide. In another embodiment, the present inventionprovides an expression vector comprising a polynucleotide encoding aCTP-modified polypeptide consisting of a Factor VIIa (FVIIa) polypeptideand three to five gonadotropin carboxy terminal peptides (CTPs) attachedto the carboxy terminus of said FVIIa polypeptide.

In another embodiment, the invention provides a cell comprising theexpression vector as described herein. In another embodiment, thepresent invention provides a cell comprising an expression vectorcomprising a polynucleotide encoding a CTP-modified polypeptideconsisting of a Factor IX (FIX) polypeptide and three gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FIX polypeptide. In another embodiment, the present inventionprovides a cell comprising an expression vector comprising apolynucleotide encoding a CTP-modified polypeptide consisting of aFactor VIIa (FVIIa) polypeptide and three gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of said FVIIapolypeptide.

In another embodiment, the invention provides a composition comprisingthe expression vector as described herein. In another embodiment, thepresent invention provides a composition comprising an expression vectorcomprising a polynucleotide encoding a CTP-modified polypeptideconsisting of a Factor IX (FIX) polypeptide and three gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FIX polypeptide. In another embodiment, the present inventionprovides a composition comprising an expression vector comprising apolynucleotide encoding a CTP-modified polypeptide consisting of aFactor VIIa (FVIIa) polypeptide and three gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of said FVIIapolypeptide.

In another embodiment, the invention provides a composition comprisingthe cell as described herein. In another embodiment, the cell is aeukaryotic cell. In another embodiment, the cell is a prokaryotic cell.

In another embodiment, the present invention provides a method ofproducing a CTP-modified coagulation factor, comprising the step ofattaching one to ten chorionic gonadotropin carboxy terminal peptides(CTPs) to the carboxy terminus of said coagulation factor, therebyproducing a CTP-modified coagulation factor. In another embodiment, thepresent invention provides a method of producing a CTP-modifiedcoagulation factor, comprising the step of attaching one to tenpolynucleotide sequences encoding a chorionic gonadotropin carboxyterminal peptide (CTP) to the carboxy terminus of a polynucleotidesequence encoding said coagulation factor, thereby producing aCTP-modified coagulation factor. In another embodiment, the presentinvention provides a method of producing a CTP-modified Factor IX (FIX)polypeptide, comprising the step of attaching three chorionicgonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus ofsaid FIX polypeptide, thereby producing a CTP-modified FIX polypeptide.In another embodiment, the present invention provides a method ofproducing a CTP-modified Factor VIIa (FVIIa) polypeptide, comprising thestep of attaching three chorionic gonadotropin carboxy terminal peptides(CTPs) to the carboxy terminus of said FVIIa polypeptide, therebyproducing a CTP-modified FVIIa polypeptide.

In another embodiment, the engineered coagulation factors of the presentinvention are synthesized using a polynucleotide molecule encoding apolypeptide of the present invention. In some embodiments, thepolynucleotide molecule encoding the engineered coagulation factors ofthe present invention is ligated into an expression vector, comprising atranscriptional control of a cis-regulatory sequence (e.g., promotersequence). In some embodiments, the cis-regulatory sequence is suitablefor directing constitutive expression of an engineered coagulationfactor of the present invention. In some embodiments, the cis-regulatorysequence is suitable for directing tissue-specific expression of theengineered coagulation factors of the present invention. In someembodiments, the cis-regulatory sequence is suitable for directinginducible expression of the engineered coagulation factors of thepresent invention.

In some embodiment, tissue-specific promoters suitable for use with thepresent invention include sequences which are functional in one or morespecific cell populations. Examples include, but are not limited to,promoters such as albumin that is liver-specific [Pinkert et al., (1987)Genes Dev. 1:268-277], lymphoid-specific promoters [Calame et al.,(1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cellreceptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins;[Banerji et al. (1983) Cell 33729-740], neuron-specific promoters suchas the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad.Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al.(1985) Science 230:912-916] or mammary gland-specific promoters such asthe milk whey promoter (U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Inducible promoters suitable for use with thepresent invention include, for example, the tetracycline-induciblepromoter (Srour, M. A., et al., 2003. Thromb. Haemost. 90: 398-405).

In one embodiment, the phrase “a polynucleotide molecule” refers to asingle or double stranded nucleic acid sequence which is isolated andprovided in the form of an RNA sequence, a complementary polynucleotidesequence (cDNA), a genomic polynucleotide sequence and/or a compositepolynucleotide sequences (e.g., a combination of the above).

In one embodiment, a “complementary polynucleotide sequence” refers to asequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA-dependent DNA polymerase.In one embodiment, the sequence can be subsequently amplified in vivo orin vitro using a DNA polymerase.

In one embodiment, a “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

In one embodiment, a “composite polynucleotide sequence” refers to asequence, which is at least partially complementary and at leastpartially genomic. In one embodiment, a composite sequence can includesome exonal sequences required to encode the polypeptide of the presentinvention, as well as some intronic sequences interposing therebetween.In one embodiment, the intronic sequences can be of any source,including of other genes, and typically will include conserved splicingsignal sequences. In one embodiment, intronic sequences includecis-acting expression regulatory elements.

In one embodiment, following expression and secretion, the signalpeptides are cleaved from the precursor engineered coagulation factorsresulting in the mature engineered coagulation factors.

In some embodiments, polynucleotides of the present invention areprepared using PCR techniques, or any other method or procedure known toone skilled in the art. In some embodiments, the procedure involves theligation of two different DNA sequences (See, for example, “CurrentProtocols in Molecular Biology”, eds. Ausubel et al., John Wiley & Sons,1992).

In one embodiment, polynucleotides of the present invention which encodethe engineered coagulation factors are inserted into expression vectors(i.e., a nucleic acid construct) to enable expression of the recombinantpolypeptide. In one embodiment, the expression vector of the presentinvention includes additional sequences which render this vectorsuitable for replication and integration in prokaryotes. In oneembodiment, the expression vector of the present invention includesadditional sequences which render this vector suitable for replicationand integration in eukaryotes. In one embodiment, the expression vectorof the present invention includes a shuttle vector which renders thisvector suitable for replication and integration in both prokaryotes andeukaryotes. In some embodiments, cloning vectors comprise transcriptionand translation initiation sequences (e.g., promoters, enhances) andtranscription and translation terminators (e.g., polyadenylationsignals).

In one embodiment, a variety of prokaryotic or eukaryotic cells can beused as host-expression systems to express the coagulation factors ofthe present invention. In some embodiments, these include, but are notlimited to, microorganisms, such as bacteria transformed with arecombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvector containing the polypeptide coding sequence; yeast transformedwith recombinant yeast expression vectors containing the polypeptidecoding sequence; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors,such as Ti plasmid, containing the polypeptide coding sequence.

In some embodiments, non-bacterial expression systems are used (e.g.mammalian expression systems such as CHO cells) to express thecoagulation factors of the present invention. In one embodiment, theexpression vector used to express polynucleotides of the presentinvention in mammalian cells is pCI-DHFR vector comprising a CMVpromoter and a neomycin resistance gene. Construction of the pCI-dhfrvector is described, according to one embodiment, in Example 1.

In some embodiments, in bacterial systems of the present invention, anumber of expression vectors can be advantageously selected dependingupon the use intended for the polypeptide expressed. In one embodiment,large quantities of polypeptide are desired. In one embodiment, vectorsthat direct the expression of high levels of the protein product,possibly as a fusion with a hydrophobic signal sequence, which directsthe expressed product into the periplasm of the bacteria or the culturemedium where the protein product is readily purified are desired. In oneembodiment, certain fusion proteins are engineered with a specificcleavage site to aid in recovery of the polypeptide. In one embodiment,vectors adaptable to such manipulation include, but are not limited to,the pET series of E. coli expression vectors [Studier et al., Methods inEnzymol. 185:60-89 (1990)].

In one embodiment, yeast expression systems are used. In one embodiment,a number of vectors containing constitutive or inducible promoters canbe used in yeast as disclosed in U.S. Pat. No. 5,932,447, which isincorporated by reference herein in its entirety. In another embodiment,vectors which promote integration of foreign DNA sequences into theyeast chromosome are used.

In one embodiment, the expression vector of the present invention canfurther include additional polynucleotide sequences that allow, forexample, the translation of several proteins from a single mRNA such asan internal ribosome entry site (IRES) and sequences for genomicintegration of the promoter-chimeric polypeptide.

In some embodiments, mammalian expression vectors include, but are notlimited to, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2,pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB,pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

In some embodiments, expression vectors containing regulatory elementsfrom eukaryotic viruses such as retroviruses are used in the presentinvention. SV40 vectors include pSVT7 and pMT2. In some embodiments,vectors derived from bovine papilloma virus include pBV-1MTHA, andvectors derived from Epstein Bar virus include pHEBO, and p205. Otherexemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5,baculovirus pDSVE, and any other vector allowing expression of proteinsunder the direction of the SV-40 early promoter, SV-40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

In some embodiments, recombinant viral vectors are useful for in vivoexpression of the coagulation factors of the present invention sincethey offer advantages such as lateral infection and targetingspecificity. In one embodiment, lateral infection is inherent in thelife cycle of, for example, a retrovirus and is the process by which asingle infected cell produces many progeny virions that bud off andinfect neighboring cells. In one embodiment, the result is that a largearea becomes rapidly infected, most of which was not initially infectedby the original viral particles. In one embodiment, viral vectors areproduced that are unable to spread laterally. In one embodiment, thischaracteristic can be useful if the desired purpose is to introduce aspecified gene into only a localized number of targeted cells.

In one embodiment, various methods can be used to introduce theexpression vector of the present invention into cells. Such methods aregenerally described in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Springs Harbor Laboratory, New York (1989, 1992), inAusubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRCPress, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press,Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectorsand Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at.[Biotechniques 4 (6): 504-512, 1986] and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992, incorporated herein by reference, for positive-negativeselection methods.

In some embodiments, introduction of nucleic acid by viral infectionoffers several advantages over other methods such as lipofection andelectroporation, since higher transfection efficiency can be obtaineddue to the infectious nature of viruses.

In one embodiment, it will be appreciated that the engineeredcoagulation factors of the present invention can also be expressed froma nucleic acid construct administered to the individual employing anysuitable mode of administration, described hereinabove (i.e., in vivogene therapy). In one embodiment, the nucleic acid construct isintroduced into a suitable cell via an appropriate gene deliveryvehicle/method (transfection, transduction, homologous recombination,etc.) and an expression system as needed and then the modified cells areexpanded in culture and returned to the individual (i.e., ex vivo genetherapy).

In one embodiment, plant expression vectors are used. In one embodiment,the expression of a polypeptide coding sequence is driven by a number ofpromoters. In some embodiments, viral promoters such as the 35S RNA and19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)],or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311(1987)] are used. In another embodiment, plant promoters are used suchas, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J.3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] orheat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B [Gurley etal., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructsare introduced into plant cells using Ti plasmid, Ri plasmid, plantviral vectors, direct DNA transformation, microinjection,electroporation and other techniques well known to the skilled artisan.See, for example, Weissbach & Weissbach [Methods for Plant MolecularBiology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Otherexpression systems such as insects and mammalian host cell systems,which are well known in the art, can also be used by the presentinvention.

It will be appreciated that other than containing the necessary elementsfor the transcription and translation of the inserted coding sequence(encoding the polypeptide), the expression construct of the presentinvention can also include sequences engineered to optimize stability,production, purification, yield or activity of the expressedpolypeptide.

In some embodiments, transformed cells are cultured under effectiveconditions, which allow for the expression of high amounts ofrecombinant engineered coagulation factors. In some embodiments,effective culture conditions include, but are not limited to, effectivemedia, bioreactor, temperature, pH and oxygen conditions that permitprotein production. In one embodiment, an effective medium refers to anymedium in which a cell is cultured to produce the recombinantpolypeptide of the present invention. In some embodiments, a mediumtypically includes an aqueous solution having assimilable carbon,nitrogen and phosphate sources, and appropriate salts, minerals, metalsand other nutrients, such as vitamins. In some embodiments, cells of thepresent invention can be cultured in conventional fermentationbioreactors, shake flasks, test tubes, microtiter dishes and petriplates. In some embodiments, culturing is carried out at a temperature,pH and oxygen content appropriate for a recombinant cell. In someembodiments, the determination of culturing conditions are within theexpertise of one of ordinary skill in the art.

In some embodiments, depending on the vector and host system used forproduction, resultant engineered coagulation factors of the presentinvention either remain within the recombinant cell, are secreted intothe fermentation medium, are secreted into a space between two cellularmembranes, such as the periplasmic space in E. coli; or are retained onthe outer surface of a cell or viral membrane.

In one embodiment, following a predetermined time in culture, recoveryof the recombinant engineered coagulation factor is effected.

In one embodiment, the phrase “recovering the recombinant engineeredcoagulation factor” used herein refers to collecting the wholefermentation medium containing the polypeptide and need not implyadditional steps of separation or purification.

In one embodiment, engineered coagulation factors of the presentinvention are purified using a variety of standard protein purificationtechniques, such as, but not limited to, affinity chromatography, ionexchange chromatography, filtration, electrophoresis, hydrophobicinteraction chromatography, gel filtration chromatography, reverse phasechromatography, concanavalin A chromatography, chromatofocusing anddifferential solubilization.

In one embodiment, to facilitate recovery, the expressed coding sequencecan be engineered to encode the engineered coagulation factor of thepresent invention and fused cleavable moiety. In one embodiment, afusion protein can be designed so that the polypeptide can be readilyisolated by affinity chromatography; e.g., by immobilization on a columnspecific for the cleavable moiety. In one embodiment, a cleavage site isengineered between the engineered coagulation factor and the cleavablemoiety and the polypeptide can be released from the chromatographiccolumn by treatment with an appropriate enzyme or agent thatspecifically cleaves the fusion protein at this site [e.g., see Booth etal., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem.265:15854-15859 (1990)].

In one embodiment, the engineered coagulation factor of the presentinvention is retrieved in “substantially pure” form.

In one embodiment, the phrase “substantially pure” refers to a puritythat allows for the effective use of the protein in the applicationsdescribed herein.

In one embodiment, the engineered coagulation factor of the presentinvention can also be synthesized using in vitro expression systems. Inone embodiment, in vitro synthesis methods are well known in the art andthe components of the system are commercially available.

In some embodiments, the recombinant engineered coagulation factors aresynthesized and purified; their therapeutic efficacy can be assayedeither in vivo or in vitro. In one embodiment, the binding activities ofthe recombinant engineered coagulation factors of the present inventioncan be ascertained using various assays as known to one of skill in theart.

In another embodiment, the engineered coagulation factor of the presentinvention can be provided to the individual per se. In one embodiment,the engineered coagulation factor of the present invention can beprovided to the individual as part of a pharmaceutical composition whereit is mixed with a pharmaceutically acceptable carrier.

In another embodiment, a “pharmaceutical composition” refers to apreparation of one or more of the active ingredients described hereinwith other chemical components such as physiologically suitable carriersand excipients. The purpose of a pharmaceutical composition is tofacilitate administration of a compound to an organism.

In another embodiment, “active ingredient” refers to the polypeptidesequence of interest, which is accountable for the biological effect.

In another embodiment, any of the compositions of the present inventionwill comprise at least one CTP sequence bound only to the carboxyterminus of an engineered coagulation factor of interest, in any form.In one embodiment, the present invention provides combined preparations.In one embodiment, “a combined preparation” defines especially a “kit ofparts” in the sense that the combination partners as defined above canbe dosed independently or by use of different fixed combinations withdistinguished amounts of the combination partners i.e., simultaneously,concurrently, separately or sequentially. In some embodiments, the partsof the kit of parts can then, e.g., be administered simultaneously orchronologically staggered, that is at different time points and withequal or different time intervals for any part of the kit of parts. Theratio of the total amounts of the combination partners, in someembodiments, can be administered in the combined preparation. In oneembodiment, the combined preparation can be varied, e.g., in order tocope with the needs of a patient subpopulation to be treated or theneeds of the single patient which different needs can be due to aparticular disease, severity of a disease, age, sex, or body weight ascan be readily made by a person skilled in the art.

In another embodiment, the phrases “physiologically acceptable carrier”and “pharmaceutically acceptable carrier” which are interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. In one embodiment, one of the ingredients includedin the pharmaceutically acceptable carrier can be for examplepolyethylene glycol (PEG), a biocompatible polymer with a wide range ofsolubility in both organic and aqueous media (Mutter et al. (1979)).

In another embodiment, “excipient” refers to an inert substance added toa pharmaceutical composition to further facilitate administration of anactive ingredient. In one embodiment, excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs are found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Various embodiments of dosage ranges are contemplated by this invention.The dosage of the engineered coagulation factor of the presentinvention, in one embodiment, is in the range of 0.005-100 mg/day. Inanother embodiment, the dosage is in the range of 0.005-5 mg/day. Inanother embodiment, the dosage is in the range of 0.01-50 mg/day. Inanother embodiment, the dosage is in the range of 0.1-20 mg/day. Inanother embodiment, the dosage is in the range of 0.1-10 mg/day. Inanother embodiment, the dosage is in the range of 0.01-5 mg/day. Inanother embodiment, the dosage is in the range of 0.001-0.01 mg/day. Inanother embodiment, the dosage is in the range of 0.001-0.1 mg/day. Inanother embodiment, the dosage is in the range of 0.1-5 mg/day. Inanother embodiment, the dosage is in the range of 0.5-50 mg/day. Inanother embodiment, the dosage is in the range of 0.2-15 mg/day. Inanother embodiment, the dosage is in the range of 0.8-65 mg/day. Inanother embodiment, the dosage is in the range of 1-50 mg/day. Inanother embodiment, the dosage is in the range of 5-10 mg/day. Inanother embodiment, the dosage is in the range of 8-15 mg/day. Inanother embodiment, the dosage is in a range of 10-20 mg/day. In anotherembodiment, the dosage is in the range of 20-40 mg/day. In anotherembodiment, the dosage is in a range of 60-120 mg/day. In anotherembodiment, the dosage is in the range of 12-40 mg/day. In anotherembodiment, the dosage is in the range of 40-60 mg/day. In anotherembodiment, the dosage is in a range of 50-100 mg/day. In anotherembodiment, the dosage is in a range of 1-60 mg/day. In anotherembodiment, the dosage is in the range of 15-25 mg/day. In anotherembodiment, the dosage is in the range of 5-10 mg/day. In anotherembodiment, the dosage is in the range of 55-65 mg/day.

In another embodiment, the dosage is in a range of 50-500 mg/day. Inanother embodiment, the dosage is in a range of 50-150 mg/day. Inanother embodiment, the dosage is in a range of 100-200 mg/day. Inanother embodiment, the dosage is in a range of 150-250 mg/day. Inanother embodiment, the dosage is in a range of 200-300 mg/day. Inanother embodiment, the dosage is in a range of 250-400 mg/day. Inanother embodiment, the dosage is in a range of 300-500 mg/day. Inanother embodiment, the dosage is in a range of 350-500 mg/day.

In one embodiment, the dosage is 20 mg/day. In one embodiment, thedosage is 30 mg/day. In one embodiment, the dosage is 40 mg/day. In oneembodiment, the dosage is 50 mg/day. In one embodiment, the dosage is0.01 mg/day. In another embodiment, the dosage is 0.1 mg/day. In anotherembodiment, the dosage is 1 mg/day. In another embodiment, the dosage is0.530 mg/day. In another embodiment, the dosage is 0.05 mg/day. Inanother embodiment, the dosage is 50 mg/day. In another embodiment, thedosage is 10 mg/day. In another embodiment, the dosage is 20-70 mg/day.In another embodiment, the dosage is 5 mg/day.

In one embodiment, the dosage of the CTP-modified coagulation factor is1-5 mg/day. In one embodiment, the dosage of the CTP-modifiedcoagulation factor is 1-3 mg/day. In another embodiment, the dosage ofthe CTP-modified coagulation factor is 2 mg/day.

In another embodiment, the dosage is 1-90 mg/day. In another embodiment,the dosage is 1-90 mg/2 days. In another embodiment, the dosage is 1-90mg/3 days. In another embodiment, the dosage is 1-90 mg/4 days. Inanother embodiment, the dosage is 1-90 mg/5 days. In another embodiment,the dosage is 1-90 mg/6 days. In another embodiment, the dosage is 1-90mg/week. In another embodiment, the dosage is 1-90 mg/9 days. In anotherembodiment, the dosage is 1-90 mg/11 days. In another embodiment, thedosage is 1-90 mg/14 days.

In another embodiment, the coagulation factor dosage is 10-50 mg/day. Inanother embodiment, the dosage is 10-50 mg/2 days. In anotherembodiment, the dosage is 10-50 mg/3 days. In another embodiment, thedosage is 10-50 mg/4 days. In another embodiment, the dosage is 10-50micrograms mg/5 days. In another embodiment, the dosage is 10-50 mg/6days. In another embodiment, the dosage is 10-50 mg/week. In anotherembodiment, the dosage is 10-50 mg/9 days. In another embodiment, thedosage is 10-50 mg/11 days. In another embodiment, the dosage is 10-50mg/14 days.

In another embodiment, a polypeptide comprising a coagulation factor andat least one CTP unit is formulated in an intranasal dosage form. Inanother embodiment, a polypeptide comprising a coagulation factor and atleast one CTP unit is formulated in an injectable dosage form. Inanother embodiment, a polypeptide comprising a coagulation factor and atleast one CTP unit is administered to a subject in a dose ranging from0.0001 mg to 0.6 mg. In another embodiment, a polypeptide comprising acoagulation factor and at least one CTP unit is administered to asubject in a dose ranging from 0.001 mg to 0.005 mg. In anotherembodiment, a polypeptide comprising a coagulation factor and at leastone CTP unit is administered to a subject in a dose ranging from 0.005mg to 0.01 mg. In another embodiment, a polypeptide comprising acoagulation factor and at least one CTP unit is administered to asubject in a dose ranging from 0.01 mg to 0.3 mg. In another embodiment,a polypeptide comprising a coagulation factor and at least one CTP unitis administered to a subject in a dose in a dose ranging from 0.2 mg to0.6 mg. In another embodiment, the coagulation factor is free of CTPs onits amino terminus.

In another embodiment, a polypeptide comprising a coagulation factor andat least one CTP unit is administered to a subject in a dose rangingfrom 1-100 micrograms. In another embodiment, a polypeptide comprising acoagulation factor and at least one CTP unit is administered to asubject in a dose ranging from 10-80 micrograms. In another embodiment,a polypeptide comprising a coagulation factor and at least one CTP unitis administered to a subject in a dose ranging from 20-60 micrograms. Inanother embodiment, a polypeptide comprising a coagulation factor and atleast one CTP unit is administered to a subject in a dose ranging from10-50 micrograms. In another embodiment, a polypeptide comprising acoagulation factor and at least one CTP unit is administered to asubject in a dose ranging from 40-80 micrograms. In another embodiment,a polypeptide comprising a coagulation factor and at least one CTP unitis administered to a subject in a dose ranging from 10-30 micrograms. Inanother embodiment, a polypeptide comprising a coagulation factor and atleast one CTP unit is administered to a subject in a dose ranging from30-60 micrograms.

In another embodiment, a polypeptide comprising a coagulation factor andat least one CTP unit is administered to a subject in a dose rangingfrom 0.2 mg to 2 mg. In another embodiment, a polypeptide comprising acoagulation factor and at least one CTP unit is administered to asubject in a dose ranging from 2 mg to 6 mg. In another embodiment, apolypeptide comprising a coagulation factor and at least one CTP unit isadministered to a subject in a dose ranging from 4 mg to 10 mg. Inanother embodiment, a polypeptide comprising a coagulation factor and atleast one CTP unit is administered to a subject in a dose ranging from 5mg and 15 mg.

In one embodiment, the dosage of the CTP-modified FIX comprises 50% ofthe amount of FIX administered in the recommended dosage of recombinantFIX (e.g., Benefix®, Wyeth or Mononine®, CSL Behring) to patients overthe same period of time. In one embodiment, the dosage of theCTP-modified FVIIa comprises 50% of the amount of FVIIa administered inthe recommended dosage of recombinant FVIIa (e.g., NovoSeven®) topatients over the same period of time. In one embodiment, the dosage ofthe CTP-modified FVII comprises 50% of the amount of FVII administeredin the recommended dosage of recombinant FVII to patients over the sameperiod of time. For example, if NovoSeven® is given at a dose of 90mcg/kg every two hours to a patient pre- or post-operatively (i.e., 7.65mg every two hours or 45.9 mg in six doses over a 12 hour period, for an85 kg patient), a CTP-modified coagulation factor of the presentinvention may be given at a dose that is 50% of the patient's 12-hourdose of recombinant FVIIa (i.e., at a dose of 23 mg given once over a12-hour period).

In another embodiment, the dosage of CTP-modified coagulation factor issuch that it contains 45% of the amount of the coagulation factor thanthat administered using the non-CTP-modified coagulation factor. Inanother embodiment, the dosage of CTP-modified coagulation factor issuch that it contains 10% of the amount of the coagulation factor thanthat administered using the non-CTP-modified coagulation factor. Inanother embodiment, the dosage of CTP-modified coagulation factor issuch that it contains 25% of the amount of the coagulation factor thanthat administered using the non-CTP-modified coagulation factor. Inanother embodiment, the dosage of CTP-modified coagulation factor issuch that it contains 35% of the amount of the coagulation factor thanthat administered using the non-CTP-modified coagulation factor. Inanother embodiment, the dosage of CTP-modified coagulation factor issuch that it contains 75% of the amount of the coagulation factor thanthat administered using the non-CTP-modified coagulation factor. Inanother embodiment, the dosage of CTP-modified coagulation factor issuch that it contains 100% of the amount of the coagulation factor thanthat administered using the non-CTP-modified coagulation factor.However, even if the dosage contains the same amount of coagulationfactor (e.g. FIX) as non-CTP-modified coagulation factor, it is stilladvantageous to subjects in that it will be administered less frequentlybecause of its increased half-life compared to recombinant coagulationfactors.

In another embodiment, a therapeutically effective amount of aconjugated coagulation factor is between 50-500 IU per kg body weightadministered once a day to once a week for FIX or 10μ/Kg-500μ/Kg forFVIIa. In another embodiment, a therapeutically effective amount of aconjugated coagulation factor is 150-250 IU per kg body weight,administered once a day. In another embodiment, a pharmaceuticalcomposition comprising a conjugated coagulation factor is formulated ata strength effective for administration by various means to a humanpatient.

In one embodiment, FIX is administered in an amount effective to bringcirculating Factor IX activity to 20-30 IU/dL in a subject. In anotherembodiment, FIX is administered in an amount effective to bringcirculating Factor IX activity to 25-50 IU/dL in a subject. In anotherembodiment, FIX is administered in an amount effective to bringcirculating Factor IX activity to 50-100 IU/dL in a subject. In anotherembodiment, FIX is administered in an amount effective to bringcirculating Factor IX activity to 100-200 IU/dL in a subject. In anotherembodiment, FIX is administered in an amount effective to bringcirculating Factor IX activity to 10-50 IU/dL in a subject. In anotherembodiment, FIX is administered in an amount effective to bringcirculating Factor IX activity to 20-100 IU/dL in a subject.

In one embodiment, the CTP-modified coagulation factor is administeredto a subject on a weekly basis. In another embodiment, the CTP-modifiedcoagulation factor is administered to a subject twice a week. In anotherembodiment, the CTP-modified coagulation factor is administered to asubject on a fortnightly (once every two weeks) basis. In anotherembodiment, the CTP-modified coagulation factor is administered to asubject twice a month. In another embodiment, the CTP-modifiedcoagulation factor is administered to a subject once a month. In anotherembodiment, the CTP-modified coagulation factor is administered to asubject on a daily basis. In another embodiment, the CTP-modifiedcoagulation factor is administered to a subject every two days.

In another embodiment, a polypeptide comprising a coagulation factor andat least one CTP unit is administered to a subject once every threedays. In another embodiment, a polypeptide comprising a coagulationfactor and at least one CTP unit is administered to a subject once everyfour days. In another embodiment, a polypeptide comprising a coagulationfactor and at least one CTP unit is administered to a subject once everyfive days. In another embodiment, a polypeptide comprising a coagulationfactor and at least one CTP unit is administered to a subject once everysix days. In another embodiment, a polypeptide comprising a coagulationfactor and at least one CTP unit is administered to a subject once every7-14 days. In another embodiment, a polypeptide comprising a coagulationfactor and at least one CTP unit is administered to a subject once every10-20 days. In another embodiment, a polypeptide comprising acoagulation factor and at least one CTP unit is administered to asubject once every 5-15 days. In another embodiment, a polypeptidecomprising a coagulation factor and at least one CTP unit isadministered to a subject once every 15-30 days.

In another embodiment, the methods of the invention include increasingthe compliance in the use of coagulation factor therapy, comprisingproviding to a subject in need thereof, a polypeptide comprising acoagulation factor and at least one chorionic gonadotropin carboxyterminal peptide (CTP) attached to the carboxy terminus of thecoagulation factor, thereby increasing compliance in the use ofcoagulation factor therapy.

In another embodiment, the methods of the invention include increasingthe compliance of patients afflicted with chronic illnesses that are inneed of a coagulation factor therapy. In another embodiment, the methodsof the invention enable reduction in the dosing frequency of acoagulation factor by modifying the coagulation factor with CTPs asdescribed hereinabove.

In another embodiment, the present invention provides a method ofreducing the dosing frequency of a Factor IX (FIX) polypeptide,comprising the step of attaching three chorionic gonadotropin carboxyterminal peptides (CTPs) to the carboxy terminus of said FIXpolypeptide, thereby reducing the dosing frequency of said FIXpolypeptide. In another embodiment, the present invention provides amethod of reducing the dosing frequency of a Factor VIIa (FVIIa)polypeptide, comprising the step of attaching three chorionicgonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus ofsaid FVIIa polypeptide, thereby reducing the dosing frequency of saidFVIIa polypeptide.

In another embodiment, the term compliance comprises adherence. Inanother embodiment, the methods of the invention include increasing thecompliance of patients in need of a coagulation factor therapy byreducing the frequency of administration of the coagulation factor. Inanother embodiment, reduction in the frequency of administration of thecoagulation factor is achieved due to the CTP modifications which renderthe CTP-modified coagulation factor more stable. In another embodiment,reduction in the frequency of administration of the coagulation factoris achieved as a result of increasing T½ of the coagulation factor. Inanother embodiment, reduction in the frequency of administration of thecoagulation factor is achieved as a result of increasing the clearancetime or reducing the clearance rate of the coagulation factor.

In another embodiment, the present invention provides a method ofreducing the clearance rate of a Factor IX (FIX) polypeptide, comprisingthe step of attaching three chorionic gonadotropin carboxy terminalpeptides (CTPs) to the carboxy terminus of said FIX polypeptide, therebyreducing the clearance rate of said FIX polypeptide. In anotherembodiment, the present invention provides a method of reducing theclearance rate of a Factor VIIa (FVIIa) polypeptide, comprising the stepof attaching three chorionic gonadotropin carboxy terminal peptides(CTPs) to the carboxy terminus of said FVIIa polypeptide, therebyreducing the clearance rate of said FVIIa polypeptide.

In another embodiment, reduction in the frequency of administration ofthe coagulation factor is achieved as a result of increasing the AUCmeasure of the coagulation factor.

In another embodiment, provided herein is a method of reducing thedosing frequency of a coagulation factor, comprising the step ofattaching one to ten CTPs to the carboxy terminus of the coagulationfactor, thereby reducing a dosing frequency of the coagulation factor.In another embodiment, provided herein is a method of reducing thedosing frequency of a coagulation factor, comprising the step ofattaching one to five CTPs to the carboxy terminus of the coagulationfactor, thereby reducing a dosing frequency of the coagulation factor.In another embodiment, provided herein is a method of reducing thedosing frequency of a coagulation factor, comprising the step ofattaching three CTPs to the carboxy terminus of the coagulation factor,thereby reducing a dosing frequency of the coagulation factor. Inanother embodiment, provided herein is a method of reducing the dosingfrequency of a coagulation factor, comprising the step of attachingthree to five CTPs to the carboxy terminus of the coagulation factor,thereby reducing a dosing frequency of the coagulation factor.

In another embodiment, provided herein is a method of increasingcompliance in the use of coagulation factor therapy, comprisingproviding to a subject in need thereof, a polypeptide comprising acoagulation factor and one to ten chorionic gonadotropin carboxyterminal peptides attached to the carboxy terminus of a coagulationfactor, thereby increasing compliance in the use of coagulation factortherapy. In another embodiment, provided herein is a method ofincreasing compliance in the use of coagulation factor therapy,comprising providing to a subject in need thereof, a polypeptidecomprising a coagulation factor and one to five chorionic gonadotropincarboxy terminal peptides attached to the carboxy terminus of acoagulation factor, thereby increasing compliance in the use ofcoagulation factor therapy. In another embodiment, provided herein is amethod of increasing compliance in the use of coagulation factortherapy, comprising providing to a subject in need thereof, apolypeptide comprising a coagulation factor and three chorionicgonadotropin carboxy terminal peptides attached to the carboxy terminusof a coagulation factor, thereby increasing compliance in the use ofcoagulation factor therapy. In another embodiment, provided herein is amethod of increasing compliance in the use of coagulation factortherapy, comprising providing to a subject in need thereof, apolypeptide comprising a coagulation factor and three to five chorionicgonadotropin carboxy terminal peptides attached to the carboxy terminusof a coagulation factor, thereby increasing compliance in the use ofcoagulation factor therapy.

In another embodiment, provided herein is a method of preventing ortreating a blood clotting or coagulation disorder in a subject,comprising providing to said subject a polypeptide comprising acoagulation factor and one to ten chorionic gonadotropin carboxyterminal peptides attached to the carboxy terminus of a coagulationfactor, thereby treating a blood clotting or coagulation disorder insaid subject. In another embodiment, provided herein is a method ofpreventing or treating a blood clotting or coagulation disorder in asubject, comprising providing to a subject in need thereof, apolypeptide comprising a coagulation factor and one to five chorionicgonadotropin carboxy terminal peptides attached to the carboxy terminusof a coagulation factor, thereby preventing or treating a blood clottingor coagulation disorder in said subject. In another embodiment, providedherein is a method of preventing or treating a blood clotting orcoagulation disorder in a subject, comprising providing to a subject inneed thereof, a polypeptide comprising a coagulation factor and threechorionic gonadotropin carboxy terminal peptides attached to the carboxyterminus of a coagulation factor, thereby preventing or treating a bloodclotting or coagulation disorder in said subject. In another embodiment,provided herein is a method of preventing or treating a blood clottingor coagulation disorder in a subject, comprising providing to a subjectin need thereof, a polypeptide comprising a coagulation factor and threeto five chorionic gonadotropin carboxy terminal peptides attached to thecarboxy terminus of a coagulation factor, thereby preventing or treatinga blood clotting or coagulation disorder in said subject.

In another embodiment, provided herein is a method of preventinghemophilia in a subject, comprising providing to said subject apolypeptide comprising a coagulation factor and one to ten chorionicgonadotropin carboxy terminal peptides attached to the carboxy terminusof a coagulation factor, thereby preventing hemophilia in said subject.In another embodiment, provided herein is a method of preventinghemophilia in a subject, comprising providing to a subject in needthereof, a polypeptide comprising a coagulation factor and one to fivechorionic gonadotropin carboxy terminal peptides attached to the carboxyterminus of a coagulation factor, thereby preventing hemophilia in saidsubject. In another embodiment, provided herein is a method ofpreventing hemophilia in a subject, comprising providing to a subject inneed thereof, a polypeptide comprising a coagulation factor and threechorionic gonadotropin carboxy terminal peptides attached to the carboxyterminus of a coagulation factor, thereby preventing hemophilia in saidsubject. In another embodiment, provided herein is a method ofpreventing hemophilia in a subject, comprising providing to a subject inneed thereof, a polypeptide comprising a coagulation factor and three tofive chorionic gonadotropin carboxy terminal peptides attached to thecarboxy terminus of a coagulation factor, thereby preventing hemophiliain said subject.

In another embodiment, the present invention shows that the compositionsprovided herein are surprisingly more effectively absorbed into thebloodstream after SC administration (see Examples 7-9 herein). To beable to administer FVIIa subcutaneously serves as an advantage as it canbe used for prophylactic applications. Subcutaneous injections are alsomuch easier for patients to self-inject, and are advantage when thepatients are very young and their veins are small and difficult to find.

In another embodiment, provided herein is a method of treatinghemophilia in a subject, comprising providing to said subject apolypeptide comprising a coagulation factor and one to ten chorionicgonadotropin carboxy terminal peptides attached to the carboxy terminusof a coagulation factor, thereby treating hemophilia in said subject. Inanother embodiment, provided herein is a method of treating hemophiliain a subject, comprising providing to a subject in need thereof, apolypeptide comprising a coagulation factor and one to five chorionicgonadotropin carboxy terminal peptides attached to the carboxy terminusof a coagulation factor, thereby treating hemophilia in said subject. Inanother embodiment, provided herein is a method of treating hemophiliain a subject, comprising providing to a subject in need thereof, apolypeptide comprising a coagulation factor and three chorionicgonadotropin carboxy terminal peptides attached to the carboxy terminusof a coagulation factor, thereby treating hemophilia in said subject. Inanother embodiment, provided herein is a method of treating hemophiliain a subject, comprising providing to a subject in need thereof, apolypeptide comprising a coagulation factor and three to five chorionicgonadotropin carboxy terminal peptides attached to the carboxy terminusof a coagulation factor, thereby treating hemophilia in said subject.

Oral administration, in one embodiment, comprises a unit dosage formcomprising tablets, capsules, lozenges, chewable tablets, suspensions,emulsions and the like. Such unit dosage forms comprise a safe andeffective amount of the desired coagulation factor of the invention,each of which is in one embodiment, from about 0.7 or 3.5 mg to about280 mg/70 kg, or in another embodiment, about 0.5 or 10 mg to about 210mg/70 kg. The pharmaceutically-acceptable carriers suitable for thepreparation of unit dosage forms for peroral administration arewell-known in the art. In some embodiments, tablets typically compriseconventional pharmaceutically-compatible adjuvants as inert diluents,such as calcium carbonate, sodium carbonate, mannitol, lactose andcellulose; binders such as starch, gelatin and sucrose; disintegrantssuch as starch, alginic acid and croscarmelose; lubricants such asmagnesium stearate, stearic acid and talc. In one embodiment, glidantssuch as silicon dioxide can be used to improve flow characteristics ofthe powder-mixture. In one embodiment, coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. In some embodiments, theselection of carrier components depends on secondary considerations liketaste, cost, and shelf stability, which are not critical for thepurposes of this invention, and can be readily made by a person skilledin the art.

In one embodiment, the oral dosage form comprises predefined releaseprofile. In one embodiment, the oral dosage form of the presentinvention comprises an extended release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises a slow release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises an immediate release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form is formulatedaccording to the desired release profile of the pharmaceutical activeingredient as known to one skilled in the art.

Peroral compositions, in some embodiments, comprise liquid solutions,emulsions, suspensions, and the like. In some embodiments,pharmaceutically-acceptable carriers suitable for preparation of suchcompositions are well known in the art. In some embodiments, liquid oralcompositions comprise from about 0.001% to about 0.933% of the desiredcompound or compounds, or in another embodiment, from about 0.01% toabout 10%.

In some embodiments, compositions for use in the methods of thisinvention comprise solutions or emulsions, which in some embodiments areaqueous solutions or emulsions comprising a safe and effective amount ofthe compounds of the present invention and optionally, other compounds,intended for topical intranasal administration. In some embodiments, hcompositions comprise from about 0.001% to about 10.0% w/v of a subjectcompound, more preferably from about 00.1% to about 2.0, which is usedfor systemic delivery of the compounds by the intranasal route.

In another embodiment, a polypeptide comprising a coagulation factor andat least one CTP unit is injected into the muscle (intramuscularinjection). In another embodiment, a polypeptide comprising acoagulation factor and at least one CTP unit is injected below the skin(subcutaneous injection). In another embodiment, a polypeptidecomprising a coagulation factor and at least one CTP unit is injectedinto the muscle. In another embodiment, a polypeptide comprising acoagulation factor and at least one CTP unit is injected into the skin.In another embodiment, a coagulation factor as described herein isadministered via systemic administration. In another embodiment, acoagulation factor as described herein is administered by intravenousinjection. In another embodiment, administration can be parenteral,pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, transnasal, intraocular,ophthalmic, epidural, buccal, rectal, transmucosal, intestinal orparenteral delivery, including intramedullary injections as well asintrathecal or direct intraventricular administration.

In another embodiment, the preparation is administered in a local ratherthan systemic manner, for example, via injection of the preparationdirectly into a specific region of a patient's body.

In one embodiment, the route of administration may be enteral. Inanother embodiment, the route may be conjunctival, transdermal,intradermal, intra-arterial, vaginal, rectal, intratumoral, parcanceral,transmucosal, intramuscular, intravascular, intraventricular,intracranial, intra-nasal, sublingual, or a combination thereof.

In another embodiment, the pharmaceutical compositions are administeredby intravenous, intra-arterial, or intramuscular injection of a liquidpreparation. In some embodiments, liquid formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In oneembodiment, the pharmaceutical compositions are administeredintravenously, and are thus formulated in a form suitable forintravenous administration. In another embodiment, the pharmaceuticalcompositions are administered intra-arterially, and are thus formulatedin a form suitable for intra-arterial administration. In anotherembodiment, the pharmaceutical compositions are administeredintramuscularly, and are thus formulated in a form suitable forintramuscular administration.

Further, in another embodiment, the pharmaceutical compositions areadministered topically to body surfaces, and are thus formulated in aform suitable for topical administration. Suitable topical formulationsinclude gels, ointments, creams, lotions, drops and the like. Fortopical administration, the compounds of the present invention arecombined with an additional appropriate therapeutic agent or agents,prepared and applied as solutions, suspensions, or emulsions in aphysiologically acceptable diluent with or without a pharmaceuticalcarrier.

In one embodiment, pharmaceutical compositions of the present inventionare manufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

In one embodiment, pharmaceutical compositions for use in accordancewith the present invention is formulated in a conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries, which facilitate processing of the active ingredientsinto preparations which, can be used pharmaceutically. In oneembodiment, formulation is dependent upon the route of administrationchosen.

In one embodiment, injectables of the invention are formulated inaqueous solutions. In one embodiment, injectables of the invention areformulated in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiological salt buffer. In someembodiments, for transmucosal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art.

In one embodiment, the preparations described herein are formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. In some embodiments, formulations for injection are presentedin unit dosage form, e.g., in ampoules or in multidose containers withoptionally, an added preservative. In some embodiments, compositions aresuspensions, solutions or emulsions in oily or aqueous vehicles, andcontain formulatory agents such as suspending, stabilizing and/ordispersing agents.

The compositions also comprise, in some embodiments, preservatives, suchas benzalkonium chloride and thimerosal and the like; chelating agents,such as edetate sodium and others; buffers such as phosphate, citrateand acetate; tonicity agents such as sodium chloride, potassiumchloride, glycerin, mannitol and others; antioxidants such as ascorbicacid, acetylcystine, sodium metabisulfote and others; aromatic agents;viscosity adjustors, such as polymers, including cellulose andderivatives thereof; and polyvinyl alcohol and acid and bases to adjustthe pH of these aqueous compositions as needed. The compositions alsocomprise, in some embodiments, local anesthetics or other actives. Thecompositions can be used as sprays, mists, drops, and the like.

In some embodiments, pharmaceutical compositions for parenteraladministration include aqueous solutions of the active preparation inwater-soluble form. Additionally, suspensions of the active ingredients,in some embodiments, are prepared as appropriate oil or water basedinjection suspensions. Suitable lipophilic solvents or vehicles include,in some embodiments, fatty oils such as sesame oil, or synthetic fattyacid esters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions contain, in some embodiments, substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. In another embodiment, the suspensionalso contains suitable stabilizers or agents which increase thesolubility of the active ingredients to allow for the preparation ofhighly concentrated solutions.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; J. E. Diederichsand al., Pharm./nd. 56 (1994) 267-275).

In another embodiment, the pharmaceutical composition delivered in acontrolled release system is formulated for intravenous infusion,implantable osmotic pump, transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump is used (see Langer, supra;Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989).In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity tothe therapeutic target, i.e., the brain, thus requiring only a fractionof the systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990).

In some embodiments, the active ingredient is in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use. Compositions are formulated, in someembodiments, for atomization and inhalation administration. In anotherembodiment, compositions are contained in a container with attachedatomizing means.

In one embodiment, the preparation of the present invention isformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

In some embodiments, pharmaceutical compositions suitable for use incontext of the present invention include compositions wherein the activeingredients are contained in an amount effective to achieve the intendedpurpose. In some embodiments, a therapeutically effective amount meansan amount of active ingredients effective to prevent, alleviate orameliorate symptoms of disease or prolong the survival of the subjectbeing treated.

In one embodiment, determination of a therapeutically effective amountis well within the capability of those skilled in the art.

Some examples of substances which can serve aspharmaceutically-acceptable carriers or components thereof are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe Tween™ brand emulsifiers; wetting agents, such sodium laurylsulfate; coloring agents; flavoring agents; tableting agents,stabilizers; antioxidants; preservatives; pyrogen-free water; isotonicsaline; and phosphate buffer solutions. The choice of apharmaceutically-acceptable carrier to be used in conjunction with thecompound is basically determined by the way the compound is to beadministered. If the subject compound is to be injected, in oneembodiment, the pharmaceutically-acceptable carrier is sterile,physiological saline, with a blood-compatible suspending agent, the pHof which has been adjusted to about 7.4.

In addition, the compositions further comprise binders (e.g. acacia,cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropylcellulose, hydroxypropyl methyl cellulose, povidone), disintegratingagents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide,croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate),buffers (e.g., Tris-HCl., acetate, phosphate) of various pH and ionicstrength, additives such as albumin or gelatin to prevent absorption tosurfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acidsalts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate),permeation enhancers, solubilizing agents (e.g., glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite,butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose,hyroxypropylmethyl cellulose), viscosity increasing agents(e.g.carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum),sweeteners (e.g. aspartame, citric acid), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid,magnesium stearate, polyethylene glycol, sodium lauryl sulfate),flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethylphthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers orpoloxamines), coating and film forming agents (e.g. ethyl cellulose,acrylates, polymethacrylates) and/or adjuvants.

Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, cellulose (e.g. Avicel™, RC-591), tragacanth and sodiumalginate; typical wetting agents include lecithin and polyethylene oxidesorbitan (e.g. polysorbate 80). Typical preservatives include methylparaben and sodium benzoate. In another embodiment, peroral liquidcompositions also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

The compositions also include incorporation of the active material intoor onto particulate preparations of polymeric compounds such aspolylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts, or spheroplasts.) Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance.

Also comprehended by the invention are particulate compositions coatedwith polymers (e.g. poloxamers or poloxamines) and the compound coupledto antibodies directed against tissue-specific receptors, ligands orantigens or coupled to ligands of tissue-specific receptors.

In some embodiments, compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. Inanother embodiment, the modified compounds exhibit substantially longerhalf-lives in blood following intravenous injection than do thecorresponding unmodified compounds. In one embodiment, modificationsalso increase the compound's solubility in aqueous solution, eliminateaggregation, enhance the physical and chemical stability of thecompound, and greatly reduce the immunogenicity and reactivity of thecompound. In another embodiment, the desired in vivo biological activityis achieved by the administration of such polymer-compound abducts lessfrequently or in lower doses than with the unmodified compound.

In some embodiments, preparation of effective amount or dose can beestimated initially from in vitro assays. In one embodiment, a dose canbe formulated in animal models and such information can be used to moreaccurately determine useful doses in humans.

In one embodiment, toxicity and therapeutic efficacy of the activeingredients described herein can be determined by standardpharmaceutical procedures in vitro, in cell cultures or experimentalanimals. In one embodiment, the data obtained from these in vitro andcell culture assays and animal studies can be used in formulating arange of dosage for use in human. In one embodiment, the dosages varydepending upon the dosage form employed and the route of administrationutilized. In one embodiment, the exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. [See e.g., Fingl, et al., (1975) “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1].

In one embodiment, depending on the severity and responsiveness of thecondition to be treated, dosing can be of a single or a plurality ofadministrations, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

In one embodiment, the amount of a composition to be administered will,of course, be dependent on the subject being treated, the severity ofthe affliction, the manner of administration, the judgment of theprescribing physician, etc.

In one embodiment, compositions including the preparation of the presentinvention formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

In another embodiment, a coagulation factor as described herein islyophilized (i.e., freeze-dried) preparation in combination with complexorganic excipients and stabilizers such as nonionic surface activeagents (i.e., surfactants), various sugars, organic polyols and/or humanserum albumin. In another embodiment, a pharmaceutical compositioncomprises a lyophilized coagulation factor as described in sterile waterfor injection. In another embodiment, a pharmaceutical compositioncomprises a lyophilized coagulation factor as described in sterile PBSfor injection. In another embodiment, a pharmaceutical compositioncomprises a lyophilized coagulation factor as described in sterile 0.9%NaCl for injection.

In another embodiment, the pharmaceutical composition comprises acoagulation factor as described herein and complex carriers such ashuman serum albumin, polyols, sugars, and anionic surface activestabilizing agents. In another embodiment, the pharmaceuticalcomposition comprises a coagulation factor as described herein andlactobionic acid and an acetate/glycine buffer. In another embodiment,the pharmaceutical composition comprises a coagulation factor asdescribed herein and amino acids, such as arginine or glutamate thatincrease the solubility of interferon compositions in water. In anotherembodiment, the pharmaceutical composition comprises a lyophilizedcoagulation factor as described herein and glycine or human serumalbumin (HSA), a buffer (e.g. acetate) and an isotonic agent (e.g NaCl).In another embodiment, the pharmaceutical composition comprises alyophilized coagulation factor as described herein and phosphate buffer,glycine and HSA.

In another embodiment, the pharmaceutical composition comprising acoagulation factor as described herein is stabilized when placed inbuffered solutions having a pH between about 4 and 7.2. In anotherembodiment, the pharmaceutical composition comprising a coagulationfactor is in a buffered solution having a pH between about 4 and 8.5. Inanother embodiment, the pharmaceutical composition comprising acoagulation factor is in a buffered solution having a pH between about 6and 7. In another embodiment, the pharmaceutical composition comprisinga coagulation factor is in a buffered solution having a pH of about 6.5.In another embodiment, the pharmaceutical composition comprising acoagulation factor as described herein is stabilized with an amino acidas a stabilizing agent and in some cases a salt (if the amino acid doesnot contain a charged side chain).

In another embodiment, the pharmaceutical composition comprising acoagulation factor as described herein is a liquid compositioncomprising a stabilizing agent at between about 0.3% and 5% by weightwhich is an amino acid.

In another embodiment, the pharmaceutical composition comprising acoagulation factor as described herein provides dosing accuracy andproduct safety. In another embodiment, the pharmaceutical compositioncomprising a coagulation factor as described herein provides abiologically active, stable liquid formulation for use in injectableapplications. In another embodiment, the pharmaceutical compositioncomprises a non-lyophilized coagulation factor as described herein.

In another embodiment, the pharmaceutical composition comprising acoagulation factor as described herein provides a liquid formulationpermitting storage for a long period of time in a liquid statefacilitating storage and shipping prior to administration.

In another embodiment, the pharmaceutical composition comprising acoagulation factor as described herein comprises solid lipids as matrixmaterial. In another embodiment, the injectable pharmaceuticalcomposition comprising a coagulation factor as described hereincomprises solid lipids as matrix material. In another embodiment, theproduction of lipid microparticles by spray congealing was described bySpeiser (Speiser and al., Pharm. Res. 8 (1991) 47-54) followed by lipidnanopellets for peroral administration (Speiser EP 0167825 (1990)). Inanother embodiment, lipids, which are used, are well tolerated by thebody (e.g. glycerides composed of fatty acids which are present in theemulsions for parenteral nutrition).

In another embodiment, the pharmaceutical composition comprising acoagulation factor as described herein comprises polymericmicroparticles. In another embodiment, the pharmaceutical compositioncomprising a coagulation factor as described herein comprisesnanoparticles. In another embodiment, the pharmaceutical compositioncomprising a coagulation factor as described herein comprises liposomes.In another embodiment, the pharmaceutical composition comprising acoagulation factor as described herein comprises lipid emulsion. Inanother embodiment, the pharmaceutical composition comprising acoagulation factor as described herein comprises microspheres. Inanother embodiment, the pharmaceutical composition comprising acoagulation factor as described herein comprises lipid nanoparticles. Inanother embodiment, the pharmaceutical composition comprising acoagulation factor as described herein comprises lipid nanoparticlescomprising amphiphilic lipids. In another embodiment, the pharmaceuticalcomposition comprising a coagulation factor as described hereincomprises lipid nanoparticles comprising a drug, a lipid matrix and asurfactant. In another embodiment, the lipid matrix has a monoglyceridecontent which is at least 50% w/w.

In one embodiment, compositions of the present invention are presentedin a pack or dispenser device, such as an FDA approved kit, whichcontain one or more unit dosage forms containing the active ingredient.In one embodiment, the pack, for example, comprise metal or plasticfoil, such as a blister pack. In one embodiment, the pack or dispenserdevice is accompanied by instructions for administration. In oneembodiment, the pack or dispenser is accommodated by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor human or veterinary administration. Such notice, in one embodiment,is labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert.

In one embodiment, it will be appreciated that the coagulation factorsof the present invention can be provided to the individual withadditional active agents to achieve an improved therapeutic effect ascompared to treatment with each agent by itself. In another embodiment,measures (e.g., dosing and selection of the complementary agent) aretaken to avoid adverse side effects which are associated withcombination therapies.

In another embodiment, the present invention provides a CTP-modifiedFactor VIIa (FVIIa) polypeptide consisting of a FVIIa polypeptide andfive gonadotropin carboxy terminal peptides (CTPs) attached to thecarboxy terminus of said FVIIa.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising a CTP-modified Factor VIIa (FVIIa) polypeptideconsisting of a FVIIa polypeptide and five gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of said FVIIa.

In another embodiment, the present invention provides a polynucleotideencoding a CTP-modified polypeptide consisting of a Factor VIIa (FVIIa)polypeptide and three gonadotropin carboxy terminal peptides (CTPs)attached to the carboxy terminus of said FVIIa polypeptide.

In another embodiment, the present invention provides an expressionvector comprising a polynucleotide encoding a CTP-modified polypeptideconsisting of a Factor VIIa (FVIIa) polypeptide and three gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid FVIIa polypeptide.

In another embodiment, the present invention provides a cell comprisingan expression vector comprising a polynucleotide encoding a CTP-modifiedpolypeptide consisting of a Factor VIIa (FVIIa) polypeptide and threegonadotropin carboxy terminal peptides (CTPs) attached to the carboxyterminus of said FVIIa polypeptide.

In another embodiment, the present invention provides a compositioncomprising an expression vector comprising a polynucleotide encoding aCTP-modified polypeptide consisting of a Factor VIIa (FVIIa) polypeptideand three gonadotropin carboxy terminal peptides (CTPs) attached to thecarboxy terminus of said FVIIa polypeptide.

In another embodiment, the present invention provides a method ofextending the biological half-life of a Factor VIIa (FVIIa) polypeptide,comprising the step of attaching three chorionic gonadotropin carboxyterminal peptides (CTPs) to the carboxy terminus of said FVIIapolypeptide, thereby extending the biological half-life of said FVIIapolypeptide.

In another embodiment, the present invention provides a method ofimproving the area under the curve (AUC) of a Factor VIIa (FVIIa)polypeptide, comprising the step of attaching three chorionicgonadotropin carboxy terminal peptides (CTPs) to the carboxy terminus ofsaid FVIIa polypeptide, thereby improving the AUC of said FVIIapolypeptide.

In another embodiment, the present invention provides a method ofreducing the dosing frequency of a Factor VIIa (FVIIa) polypeptide,comprising the step of attaching three chorionic gonadotropin carboxyterminal peptides (CTPs) to the carboxy terminus of said FVIIapolypeptide, thereby reducing the dosing frequency of said FVIIapolypeptide.

In another embodiment, the present invention provides a method ofreducing the clearance rate of a Factor VIIa (FVIIa) polypeptide,comprising the step of attaching three chorionic gonadotropin carboxyterminal peptides (CTPs) to the carboxy terminus of said FVIIapolypeptide, thereby reducing the clearance rate of said FVIIapolypeptide.

In another embodiment, the present invention provides a method ofproducing a CTP-modified Factor VIIa (FVIIa) polypeptide, comprising thestep of attaching three chorionic gonadotropin carboxy terminal peptides(CTPs) to the carboxy terminus of said FVIIa polypeptide, therebyproducing a CTP-modified FVIIa polypeptide.

In another embodiment, the present invention provides a method oftreating hemophilia in a subject comprising administering a CTP-modifiedFactor VIIa (FVIIa) polypeptide comprising a FVIIa polypeptide and threechorionic gonadotropin carboxy terminal peptides (CTPs) attached to thecarboxy terminus of said FVIIa polypeptide to said subject, therebytreating hemophilia in said subject.

In one embodiment, the present invention provides a CTP-modified FactorIX (FIX) polypeptide consisting of a FIX polypeptide and threegonadotropin carboxy terminal peptides (CTPs) attached to the carboxyterminus of said CTP-modified FIX polypeptide. In another embodiment,the present invention provides a CTP-modified FIX polypeptide, whereinthe sequence of said CTP-modified FIX polypeptide is the sequence setforth in SEQ ID NO: 31. In another embodiment, the present inventionprovides a CTP-modified FIX polypeptide, wherein at least one CTP isencoded by an amino acid sequence selected from the group consisting of:SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, the presentinvention provides a CTP-modified FIX polypeptide, wherein at least oneCTP is glycosylated. In another embodiment, the present inventionprovides a CTP-modified FIX polypeptide, wherein at least one CTP istruncated. In another embodiment, the present invention provides aCTP-modified FIX polypeptide, wherein at least one CTP is attached tosaid FIX polypeptide via a linker. In another embodiment, the presentinvention provides a CTP-modified FIX polypeptide, wherein said linkeris a peptide bond.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising the CTP-modified FIX polypeptide.

In one embodiment, the present invention provides a polynucleotideencoding a CTP-modified polypeptide consisting of a Factor IX (FIX)polypeptide and three gonadotropin carboxy terminal peptides (CTPs)attached to the carboxy terminus of said FIX polypeptide. In anotherembodiment, the present invention provides a polynucleotide, wherein thesequence of said polynucleotide is as set forth in SEQ ID NO: 30. Inanother embodiment, the present invention provides a polynucleotide,wherein at least one CTP is encoded by an amino acid sequence selectedfrom the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2. In anotherembodiment, the present invention provides a polynucleotide, wherein atleast one CTP is glycosylated. In another embodiment, the presentinvention provides a polynucleotide, wherein at least one CTP istruncated. In another embodiment, the present invention provides apolynucleotide, wherein at least one CTP is attached to said FIXpolypeptide via a linker. In another embodiment, the present inventionprovides a polynucleotide, wherein said linker is a peptide bond. Anexpression vector comprising the polynucleotide.

In one embodiment, the present invention provides a cell comprising theexpression vector.

In one embodiment, the present invention provides a compositioncomprising the expression vector.

In one embodiment, the present invention provides a method of extendingthe biological half-life of a Factor IX (FIX) polypeptide, comprisingthe step of attaching three chorionic gonadotropin carboxy terminalpeptides (CTPs) to the carboxy terminus of said FIX polypeptide, therebyextending the biological half-life of said FIX polypeptide. In anotherembodiment, the present invention provides a method, wherein at leastone CTP is encoded by an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, thepresent invention provides a method, wherein at least one CTP isglycosylated. In another embodiment, the present invention provides amethod, wherein at least one CTP is truncated. In another embodiment,the present invention provides a method, wherein at least one CTP isattached to said FIX polypeptide via a linker. In another embodiment,the present invention provides a method, wherein said linker is apeptide bond.

In one embodiment, the present invention provides a method of improvingthe area under the curve (AUC) of a Factor IX (FIX) polypeptide,comprising the step of attaching three chorionic gonadotropin carboxyterminal peptides (CTPs) to the carboxy terminus of said FIXpolypeptide, thereby improving the AUC of said FIX polypeptide. Inanother embodiment, the present invention provides a method, wherein atleast one CTP is encoded by an amino acid sequence selected from thegroup consisting of: SEQ ID NO: 1 and SEQ ID NO: 2. In anotherembodiment, the present invention provides a method, wherein at leastone CTP is glycosylated. In another embodiment, the present inventionprovides a method, wherein at least one CTP is truncated. In anotherembodiment, the present invention provides a method, wherein at leastone CTP is attached to said FIX polypeptide via a linker. In anotherembodiment, the present invention provides a method, wherein said linkeris a peptide bond.

In one embodiment, the present invention provides a method of reducingthe dosing frequency of a Factor IX (FIX) polypeptide, comprising thestep of attaching three chorionic gonadotropin carboxy terminal peptides(CTPs) to the carboxy terminus of said FIX polypeptide, thereby reducingthe dosing frequency of said FIX polypeptide. In another embodiment, thepresent invention provides a method, wherein at least one CTP is encodedby an amino acid sequence selected from the group consisting of: SEQ IDNO: 1 and SEQ ID NO: 2. In another embodiment, the present inventionprovides a method, wherein at least one CTP is glycosylated. In anotherembodiment, the present invention provides a method, wherein at leastone CTP is truncated. In another embodiment, the present inventionprovides a method, wherein at least one CTP is attached to said FIXpolypeptide via a linker. In another embodiment, the present inventionprovides a method, wherein said linker is a peptide bond.

In one embodiment, the present invention provides a method of reducingthe clearance rate of a Factor IX (FIX) polypeptide, comprising the stepof attaching three chorionic gonadotropin carboxy terminal peptides(CTPs) to the carboxy terminus of said FIX polypeptide, thereby reducingthe clearance rate of said FIX polypeptide. In another embodiment, thepresent invention provides a method, wherein at least one CTP is encodedby an amino acid sequence selected from the group consisting of: SEQ IDNO: 1 and SEQ ID NO: 2. In another embodiment, the present inventionprovides a method, wherein at least one CTP is glycosylated. In anotherembodiment, the present invention provides a method, wherein at leastone CTP is truncated. In another embodiment, the present inventionprovides a method, wherein at least one CTP is attached to said FIXpolypeptide via a linker. In another embodiment, the present inventionprovides a method, wherein at least one CTP is attached to said FVIIpolypeptide via a linker. In another embodiment, the present inventionprovides a method, wherein said linker is a peptide bond.

In one embodiment, the present invention provides a method of producinga CTP-modified Factor IX (FIX) polypeptide, comprising the step ofattaching three chorionic gonadotropin carboxy terminal peptides (CTPs)to the carboxy terminus of said FIX polypeptide, thereby producing aCTP-modified FIX polypeptide. In another embodiment, the presentinvention provides a method, wherein the sequence of said CTP-modifiedFIX polypeptide is the sequence set forth in SEQ ID NO: 31. In anotherembodiment, the present invention provides a method, wherein at leastone CTP is encoded by an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, thepresent invention provides a method, wherein at least one CTP isglycosylated. In another embodiment, the present invention provides amethod, wherein at least one CTP is truncated. In another embodiment,the present invention provides a method, wherein at least one CTP isattached to said FIX polypeptide via a linker. In another embodiment,the present invention provides a method, wherein said linker is apeptide bond.

In one embodiment, the present invention provides a method of treatinghemophilia in a subject comprising administering a CTP-modified FactorIX (FIX) polypeptide comprising a FIX polypeptide and three chorionicgonadotropin carboxy terminal peptides (CTPs) attached to the carboxyterminus of said FIX polypeptide to said subject, thereby treatinghemophilia in said subject. In another embodiment, the present inventionprovides a method, wherein the sequence of said CTP-modified FIXpolypeptide is the sequence set forth in SEQ ID NO: 31. In anotherembodiment, the present invention provides a method, wherein at leastone CTP is encoded by an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 1 and SEQ ID NO: 2. In another embodiment, thepresent invention provides a method, wherein at least one CTP isglycosylated. In another embodiment, the present invention provides amethod, wherein at least one CTP is truncated. In another embodiment,the present invention provides a method, wherein at least one CTP isattached to said FIX polypeptide via a linker. In another embodiment,the present invention provides a method, wherein said linker is apeptide bond.

As is generally known in the art, the modified peptides and proteins ofthe invention may be coupled to labels, drugs, targeting agents,carriers, solid supports, and the like, depending on the desiredapplication. The labeled forms of the modified biologicals may be usedto track their metabolic fate; suitable labels for this purpose include,especially, radioisotope labels such as iodine 131, technetium 99,indium 111, and the like. The labels may also be used to mediatedetection of the modified proteins or peptides in assay systems; in thisinstance, radioisotopes may also be used as well as enzyme labels,fluorescent labels, chromogenic labels, and the like. The use of suchlabels is particularly helpful if the peptide or protein is itself atargeting agent such as an antibody or a receptor ligand.

Similar linking techniques, along with others, may be employed to couplethe modified peptides and proteins of the invention to solid supports.When coupled, these modified peptides and proteins can then be used asaffinity reagents for the separation of desired components with whichspecific reaction is exhibited.

Finally, the modified peptides and proteins of the invention may be usedto generate antibodies specifically immunoreactive with these newcompounds. These antibodies are useful in a variety of diagnostic andtherapeutic applications, depending on the nature of the biologicalactivity of the unmodified peptide or protein. It is to be understoodthat the invention provides antibodies that are immunoreactive withCTP-modified FIX, FVII, or FVIIa as described herein. In one embodiment,such antibodies may be used to distinguish or identify CTP-modifiedcoagulation factors that were administered from endogenous coagulationfactors. In another embodiment, the antibodies may be used to localizeadministered CTP-modified coagulation factors.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference. Other general references are provided throughout thisdocument.

Example 1 Generation and Utilization of Coagulation Factor IX

Cloning and Expression of Recombinant FIX Molecule:

Factor IX clones were constructed in our eukaryotic expression vectorpCI-neo (Promega, catalog no. E1841). ORF Clone of Homo sapienscoagulation factor IX was ordered from “OriGene” (RC219065). Primerswere ordered from Sigma-Genosys.

Construction of 301-1-pCI-neo-p200-11 (Factor IX-ctp x2):

Primer 101: (SEQ ID NO: 36) 5′ GTTTAGTGAACCGTCAGAAT 3′ Primer 103^(R):(SEQ ID NO: 37) 5′ TTGAGGAAGATGTTCGTGTA 3′ (contains the SspI site offactor IX)

A PCR reaction was conducted with primer 101 and primer 103^(R) andplasmid DNA, cDNA clone of Factor IX (OriGene” RC219065) as a template;as a result of the PCR amplification, a ˜1085 bp (per 10) product wasformed and purified from the gel (the fragment containing the aminoterminus of Factor IXsequence).

Primer 98: (SEQ ID NO: 38) 5′ ATTACAGTTGTCGCAGGTGA 3′ Primer 99^(R):(SEQ ID NO: 39) 5′ GCTGGAGCTAGTGAGCTTTGTTTTTTCCTT 3′ Primer 100: (SEQ IDNO: 40) 5′ GCTCACTAGCTCCAGCAGCAAGGCC 3′ Primer 27^(R): (SEQ ID NO: 41)5′ TTTTCACTGCATTCTAGTTGTGG 3′

Three PCR reactions were performed. The first reaction was conductedwith primer 98 and primer 99^(R) and plasmid DNA, cDNA clone of FactorIX (OriGene”,RC219065) as a template; as a result of the PCRamplification, a ˜540 bp product was formed.

The second reaction was conducted with primer 100 and primer 27^(R) andplasmid DNA of 402-2-p′72-3 (hGH-CTP-CTP) as a template; as a result ofthe PCR amplification, a ˜258 bp product was formed.

The last reaction (per 3) was conducted with primers 98 and 27^(R) and amixture of the products of the previous two reactions as a template; asa result of the PCR amplification, a ˜790 bp product was formed andligated into TA cloning vector (Invitrogen, catalog K2000-01).SspI-EcoRI fragment was isolated (TA 3-3).

Another PCR reaction was conducted (per 12) with primer 101 and primer27^(R) and a mixture of the products of per 10 and SspI-EcoRI fragmentfrom per 3 as a template; as a result of the PCR amplification, a ˜1700bp product was formed (Factor IX-ctp-ctp) and ligated into TA cloningvector (Invitrogen, catalog K2000-01) (lig 180).

A mistake was found in the Factor IXsequence so fragments were replacedin order to form an insert of Factor IX-ctp-ctp with the correct DNAsequence.

TA-per 3-3 was digested with SspI and XbaI and the large fragment wasisolated (vector). TA 180-4 was digested with SspI and XbaI and thesmall fragment (insert) was isolated and ligated to the isolated largefragment of TA-per-3-3 digested with SspI and XbaI. The new plasmidTA-183-2 was digated with Sal I and NotI, and the Factor IX-CTP-CTPinsert was isolated (˜1575 bp). This fragment was inserted intoeukaryotic expression vector pCI-neo (digested with Sal I and Not I) toyield the 301-2-p200-11 clone.

pCI-dhfr-Factor 9-ctpx2 (p223-4) Construction:

Vector pCI-dhfr (p6-1) was digested with SmaI and NotI. FactorIX-CTP-CTP (p200-11) was digested with ASisI F.I. and NotI. The twofragments were ligated.

pCI-dhfr Factor 9-ctp x3 (p225-7) Construction:

Vector pCI-dhfr OXM-CTPx3 (p216-4) was digested with XbaI and ApaI.Factor IX-CTP-CTP (223-4) was digested with XbaI and ApaI. The twofragments were ligated.

pCI-dhfr Factor 9-ctp x3 T148A (p243-2) Construction:

Plasmid p225-7 contained Threonine at position 148, since the morecommon version of FIX contains Alanine at this position, Thr wasreplaced to Ala using site directed mutagenesis method.

Primer 75: (SEQ ID NO: 42) ctcccagttcaattacagct Primer 122r: (SEQ ID NO:43) ggaaaaactgcctcagcacgggtgagc Primer 123: (SEQ ID NO: 44)gtgctgaggcagtttttcctgatgtggactat Primer 124r: (SEQ ID NO: 45)caacacagtgggcagcag

Three PCR reactions were performed. The first reaction was conductedwith primer 75 and primer 122r and plasmid DNA p225-7 as a template; asa result of the PCR amplification, a ˜692 bp product was formed andpurified from the gel. A second PCR reaction was conducted with primer123 and primer 124r and plasmid DNA p225-7 as a template; as a result ofthe PCR amplification, a ˜237 bp product was formed and purified fromthe gel. The third-overlap PCR reaction was conducted with primers 75and 124r, and a mixture of the products of the previous two reactions asa template; as a result of the PCR amplification, a ˜910 bp product wasformed. This overlap PCR product was digested with XbaI and NsiI and religated into p225-7 plasmid (digested with XbaI and NsiI) to yieldFactor IX-ctpx3 T148A designated p243-2.

FIX-4CTP (p259-4) Construction:

3.5CTP fragment was isolated from oxym-4CTP (p254-3) by restrictionenzymes ApaI and XbaI. FIX+0.5CTP fragment was isolated from FIX-3CTP(p243-2) with restriction enzymes ApaI and XbaI. The two fragments wereligated.

FIX-5CTP (p260-18) Construction:

4.5CTP fragment was isolated from oxym-5CTP (255-1) by restrictionenzymes ApaI and XbaI. FIX+0.5CTP fragment was isolated from FIX-3CTP(p243-2) using enzymes ApaI and XbaI. The two fragments were ligated.

Dg44 cells were plated in 100 mm tissue culture dishes and grown to50-60% confluence. A total of 2 μg (microgram) of FIX cDNA was used forthe transfection of one 100 mm plate using the FuGene reagent (Roche) inprotein-free medium (Invitrogene CD Dg44). The media was removed 48hours after transfection and replaced with a protein-free medium(Invitrogene CD Dg44) without nucleosides and in the presence of 800μg/ml of G418 (Neomycin). After 14 days, the transfected cell populationwas transferred into T25 tissue culture flasks, and selection continuedfor an additional 10-14 days until the cells began to grow as stableclones. High expressing clones were selected. Approximately 2×10⁷ cellswere used to inoculate 300 ml of growth medium in a 1700 cm² rollerbottle (Corning, Corning N.Y.) supplemented with 5 ng/ml of Vitamin K3(menadione sodium bisulfate; Sigma). The production medium (harvest) wascollected after a rapid decrease in cell viability to about 70%. Theproduction medium was first clarified and then concentratedapproximately 20-fold and dialyzed with PBS using flow filtrationcassette (10 KDa MWCO; Millipore Corp.).

Determination of FIX Antigen Level:

FIX-CTP harvest antigen levels were determined using AssayMax Human FIXELISA kit (AssayPro-EF1009-1). The calculated protein concentration isthe average of three different dilutions in two independent runs (FIG.1A, Table 1).

TABLE 1 Calculated protein concentration FIX-CTP FIX-CTP-CTP FIX Aglevel (μg/ml) 41.9 19.2 SD 8.76 3.67 % CV 20.92 19.15

FIX SDS-PAGE—Immune Blot:

FIX-CTP harvests or purified rhFIX (American Diagnostics), 100 ng ofprotein, were loaded on 12% Tris-Glycine gel using Precision Plus DualColor Protein Marker (Bio-Rad). The SDS-PAGE analysis was performed byWestern immunoblot using anti-human FIX polyclonal antibody andanti-human gamma carboxylation monoclonal antibody (AmericanDiagnostics). As previously reported, rhFIX migrated at 55 KDa, whileFIX fused to two CTPs migrated at 75 KDa. Both variants of FIX-CTPproteins were shown to be gamma carboxylated, an essentialpost-translation modification for FIX activity and function (FIG. 1B).

Determination of FIX Chromogenic Activity:

A comparative assessment of the in vitro potency of FIX-CTP harvestsversus rhFIX protein (American Diagnostics) was performed using thecommercially available chromogenic activity test kit, BIOPHEN (HyphenBioMed 221802). In the presence of thrombin, phospholipids, calcium,excess amounts of FXIa activates sampled FIX into FIXa. FIXa forms anenzymatic complex with thrombin, activated FVIII:C (supplied in anexcess amounts), phospholipids, and calcium and activates Factor X,present in the assay system, into FXa. The activity directly correlateswith the amount of FIX, which is the limiting factor. The generated FXais then measured by its specific activity on FXa chromogenic substrate(pNA). The amount of pNA generated is directly proportional to FIXaactivity. rhFIX and FIX-CTP harvests were serially diluted, and thepotency was assessed by comparing a dose-response curve of the FIXharvests to a reference preparation consisting of rhFIX or human plasma.The average EC50 of FIX was 21 ng/ml, while the FIX-(CTP)₂ harvestcalculated EC50 was 382 ng/ml, and the FIX-CTP harvest calculated EC50was 1644 ng/ml. An approximately 15-fold decrease in the enzymaticactivity of the FIX-(CTP)₂ harvest was observed (FIG. 2).

FIX Clotting Activity (aPTT):

The activated partial thromboplastin time (aPTT) is a measure of theintegrity of the intrinsic and common pathways of the coagulationcascade. The aPTT is the time, in seconds, for plasma to clot followingthe addition of an intrinsic pathway activator, phospholipid andcalcium. The aPTT reagent is called a partial thromboplastin becausetissue factor is not included with the phospholipid as it is with theprotime (PT) reagent. The activator initiates the system and then theremaining steps of the intrinsic pathway take place in the presence ofphospholipid. Reference aPTT range varies from laboratory to laboratory,but is usually in the range of 27-34 seconds.

The principal of the assay was to quantitate the ability of FIX-CTPharvests to restore the clotting activity of FIX-depleted human plasmaby the addition of rhFIX. 300 μl of FIX-deficient human plasma was mixedwith 100 μl of rhFIX or FIX-CTP harvests and serially diluted. Followinga 60 second incubation at 37° C., thromboplastin, CaCl₂, andphospholipids were added to the mixture, and clotting time in secondswas determined (performed by American Medical Laboratories). The potencywas assessed by comparing a dose-response curve of the FIX harvests to areference preparation consisting of rhFIX or human plasma. One unit ofFIX activity corresponds to the FIX concentration that equals theactivity of one ml normal human plasma. The presented aPTT resultsindicate that FIX-(CTP)₂ exhibit a 5.7-fold reduction in its specificcoagulation activity compared to rhFIX (Table 2). Moreover, the aPTTresults together with the chromogenic activity in vitro assay suggestthat FIX-(CTP)₂ harvest has an improved enzymatic activity vs. FIX-CTPharvest (Table 2). An improved activity of FIX-CTP proteins can beobtained following optimization of the expression system (i.e.co-transfection with Furin and optimization of Vitamin K3 mediumconcentration), which was strengthened following super-transfection withFurin (data not shown).

TABLE 2 FIX clotting activity rhFIX(AD) FIX-CTP FIX-CTP-CTP PTT (μg/ml)PTT (Sec) (μg/ml) PTT (Sec) (μg/ml) (Sec) 5 31.3 9 45.2 4 47.5 1.25 35.72.25 53.3 1 55.9 0.3125 43 0.5625 64.1 0.25 67 0.078125 52.1 0.14062576.3 0.0625 77.4

Pharmacokinetic Study:

rhFIX (American Diagnostic) and FIX-CTP harvests were administered in asingle intravenous injection to Sprague-Dawley rats (six rats persubstance) at a dose of 75 μg/kg body weight (Table 3).

TABLE 3 PK study plan of operation Dose No. of Dose Level InjectedTreated Test animals/ Dose Level (μg per Vol. Con. *Time-Points GroupsArticle group Route Gender (μg/kg) animal) (μl) (μg/ml) (hourspost-dose) 1 rFIX 6 IV M 75 15 500 30 0 (Pre-dose) 0.083, 0.5, 1.5, 4,8, 24, 48, 72. 2 rFIX- 6 IV M 75 15 500 30 0 (Pre-dose) CTP 0.083, 0.5,1.5, 4, 8, 24, 48, 72. 3 rFIX- 6 IV M 75 15 1000 15 0 (Pre-dose) CTP-0.083, 0.5, 1.5, CTP 4, 8, 24, 48, 72.

Blood samples were drawn retro-orbitally from 3 rats alternately at0.083, 0.5 1.5, 4, 8, 24, 48, and 72 hours post-dosing. Plasma wasprepared immediately after sampling and stored at −20° C. untilanalysis. FIX concentration was quantitated by FIX ELISA-specific assay(AssayPro). A pharmacokinetic profile was calculated for each proteinand represents the mean of 3 animals at each time point (FIG. 3). Theterminal half-lives were calculated using PK solutions 2.0 software.Table 4 summarizes the observed FIX concentrations at the differentsampling time points.

TABLE 4 Observed FIX concentrations Time FIX-CTP-CTP (Hr) FIX-AD (ng/ml)FIX-CTP (ng/ml) (ng/ml) 0.083 1506.7 1477.5 1914.8 0.5 1949.8 1150.11830.1 1.5 2189.4 1009.0 1264.3 4 733.90 709.33 1000.00 8 319.80 167.201234.67 24 BLQ 54.625 230 48 BLQ BLQ 120.9

The PK profile and summary of the terminal half-lives are summarized inTable 5. FIX-CTP harvests exhibit an improved T½_(β) values compared torhFIX (2- and 5-fold increases, respectively). Since in FIX dosingcollection, animal serum concentrations of FIX at 24 hr were below limitof quantitation (BLQ), additional PK parameters were not calculated.

TABLE 5 Summary of PK parameters Ratio Product Terminal half-life-(hr)(FIX-(CTP)_(X)/rhFIX) rhFIX (American 2.62 — Diagnostics) FIX-CTP 5.552.11 FIX-CTP (FIX-CTP-CTP) 12.9 4.92

In this study, a novel approach was described for prolonging FIXhalf-life while retaining the therapeutic potency. Adding a CTP peptideto an active protein has a harmful potential in interfering with theprotein's activity. Therefore, the generation of an active recombinantFIX-CTP by adding a CTP sequence at the C-terminus of the FIX isunexpected.

Characterization of an Immunoaffinity Purified FIX-CTP-CTP FIX-CTP-CTPPurification

In order to evaluate a protein at high grade content with increasedactivity whose PK profile mimics and can be extrapolated to a clinicalsetting, FIX-CTP-CTP is a FIX modified with 2 CTP units in tandem in itscarboxy-terminal. FIX-CTP-CTP was purified using matrix-bound monoclonalantibody against γ carboxyglutamyl (Gla) residues present in theN-terminal region of FIX (American Diagnostics Cat. #3570MX). Themonoclonal antibody was bound to Sepharose CL-4B. The FIX-CTP-CTPharvest at a concentration of 88 μg/ml was dialyzed against 20 mM Tris,150Mm NaCl and 10 mM EDTA at PH=7.4. The loading rate was 0.5 ml/min,elution was performed using 20Mm Tris-HCl, 350 mM NaCl and 50 mM CaCl,and the unbound fraction was recycled five times. Finally, the elutionfraction was dialyzed with PBS, pulled and concentrated.

Determination of FIX Antigen Level:

FIX-CTP harvests, FIX-(CTP)₂ harvests, and FIX-(CTP)₂ purified proteinlevels were determined using the Human FIX ELISA kit (AffinityBiologicals; Cat. #FIX-AG RUO). The calculated protein concentration(μg/ml) is the average of two independent runs (FIG. 4, Table 6).

TABLE 6 Calculated protein concentration FIX-CTP-CTP FIX-CTP FIX-CTP-CTP(purified) FIX Ag level 125.78 88.53 172.9 (μg/ml) SD 17.28 21.31 2.63 %CV 13.74 24.08 1.52

Additionally, FIX-CTP-CTP was quantitated by Bradford assay. Thecalculated concentration was 202 μg/ml, which is similar to theconcentration obtained by human FIX ELISA.

SDS-PAGE Blots:

FIX-CTP-CTP harvest, unbound fraction and purified protein, were loadedon a 12% Tris-Glycine gel using Precision Plus Dual Color Protein Marker(Bio-Rad). The SDS-PAGE Coomassie analysis was performed by staining thegel with Coommasie blue reagent (800 ng of protein). A Westernimmunoblot was performed with 100 ng of protein, anti-human FIXpolyclonal antibody (Ab), and anti-human gamma carboxylation monoclonalAb (American Diagnostics Cat #499 and #3570). The immunoaffinitypurification procedure significantly enriched the FIX-CTP-CTP portionwhile reduced impurity (FIG. 5).

N-terminal Sequencing:

FIX-CTP-CTP purified protein was separated by 12% Tris-Glycine SDS-PAGEand subsequently electro-blotted to PVDF membrane. The band of interestwas cut out and put on a purified Biobrene treated glass fiber filter.The N-terminal sequence analysis was carried out by Edmann degradationusing a pulsed liquid protein sequencer equipped with a 140 C HPLCmicro-gradient system. N-terminal sequencing revealed that FIX-CTP-CTPis a mixture of incomplete and complete pro-peptide cleaved proteins.Inadequate pro-peptide cleavage was shown to reduce FIX coagulationactivity. By co-transfection with Furin, the pro-peptide cleavageprocess can be an improved.

Determination of FIX Chromogenic Activity:

A comparative assessment of the in vitro potency of FIX-CTP-CTP purifiedprotein versus rhFIX (American Diagnostics) and a pool of human normalplasma was performed using the commercially available chromogenicactivity test kit, BIOPHEN (Hyphen BioMed 221802). In the presence ofthrombin, phospholipids and calcium, excess amounts of FXIa activatesFIX into FIXa. FIXa forms an enzymatic complex with thrombin (suppliedin excess amounts), phospholipids and calcium activates Factor X,present in the assay system, into FXa. The activity directly correlateswith the amount of FIX, which is the limiting factor. The generated FXawas measured by its specific activity on FXa chromogenic substrate(pNA). The amount of pNA generated was directly proportional to FIXaactivity. rhFIX, human plasma and FIX-CTP-CTP were serially diluted, andpotency was assessed by comparing a dose-response curve (FIG. 6). Theaverage EC₅₀ of rhFIX was 68.74 ng/ml while FIX-CTP-CTP calculated EC₅₀was 505 ng/ml. An approximately 7-fold decrease in the enzymaticactivity of FIX-CTP-CTP was observed vs. recombinant FIX and a 16.5-folddecrease versus normal human pulled plasma. This reduced activity couldbe explained by inadequate cleavage of N-terminal pro-peptide, which wasidentified by N-terminal analysis.

FIX Clotting Activity (aPTT):

The activated partial thromboplastin time (aPTT) is a measure of theintegrity of the intrinsic and common pathways of the coagulationcascade. The aPTT is the time (measured in seconds) it takes plasma toclot following the addition of an intrinsic pathway activator,phospholipid and calcium.

The assay quantitated the ability of the FIX-CTP-CTP protein to restorethe clotting activity of FIX depleted human plasma by the addition ofrhFIX. 300 μl of FIX-deficient human plasma was mixed with 100 μl ofrhFIX, FIX-CTP-CTP (FIX-CTP-CTP (the CTP are in tandem at theC-terminal)), or normal pool human plasma which was further diluted.Following a 60 second incubation at 37° C., Tissue Factor (TF), CaCl₂,and phospholipids were added to the mixture. Clotting time in secondswas determined. Potency was assessed by comparing a dose-response curveof FIX-CTP-CTP to a reference preparation of rhFIX or human plasma. Oneunit of FIX was defined as the amount of FIX which equals to theactivity of 1 ml human normal plasma.

The aPTT results indicate that FIX-CTP-CTP coagulation activity is only1.4 less than normal pool human plasma and similar to the rhFIX. TheaPTT results together with the chromogenic activity in vitro assaysuggest that FIX-CTP-CTP purification did not damage its activity.

Pharmacokinetic Activity of FIX-CTP-CTP:

Purified FIX-CTP-CTP, rhFIX (American Diagnostic) and harvestscontaining FIX-CTP-CTP and FIX-CTP were administered in a singleintravenous injection to Sprague-Dawley rats (eight rats per substance)in a dose of 100 μg/kg body weight (Table 7).

TABLE 7 PK study outline No. of Dose animals/ Dose Level InjectedTreated group/ Level (μg per Vol. Con. Time-Points Groups Test Articletime point (μg/kg) animal) (μl) (μg/ml) (hours post-dose) A rFIX 8 10020 500 40 0 (Pre-dose) 0.083, 0.5, 1, 2, 4, 7, 10, 24, 48, 72. BrFIX-CTP 8 100 20 500 40 0 (Pre-dose) (harvest) 0.083, 0.5, 1, 2, 4, 7,10, 24, 48, 72. C rFIX-CTP- 6 100 20 500 40 0 (Pre-dose) CTP(harvest)0.083, 0.5, 1, 2, 4, 7, 10, 24, 48, 72. D rFIX-CTP- 4 100 20 500 400.083, 0.5 1, 2, 4, CTP (purified) 7, 10, 24, 4, 8, 72.

Blood samples were drawn retro-orbitally from 4 rats alternately at0.083, 0.5, 2, 4, 7 10, 24, 48, and 72 hours post-dosing. Citratedplasma (0.32%) was prepared immediately after sampling and stored at−20° C. until analysis. FIX concentration was quantitated using a humanFIX ELISA kit (Affinity Biologicals). The pharmacokinetic profile wascalculated for each protein as the mean of 4 animals at each time point(FIG. 7). The terminal half-life was calculated using PK Solutions 2.0Software. Table 8 summarizes the observed FIX concentrations atdifferent sampling time points.

TABLE 8 Observed FIX concentrations Time FIX-CTP FIX-(CTP)₂ rhFIXPurified FIX- (hr) harvest ng/ml harvest ng/ml ng/ml CTP-CTP ng/ml 0.0851038.97 1123.62 325.05 886.48 0.5 939.12 956.80 274.58 670.92 1 791.97843.85 222.90 674.17 2 304.98 673.31 186.00 503.91 4 315.37 525.50109.69 357.36 7 171.45 384.36 67.62 257.02 10 50.34 250.73 40.20 158.6624 10.07 78.50 BLQ 52.13 48 BLQ 23.40 BLQ 18.07

A summary of the PK parameters are presented in Table 9.

TABLE 9 Summary of PK parameters T½ AUC MRT Vd CL (hr) ng-hr/ml (hr)ml/Kg Ml/hr/Kg FIX-CTP harvest 4.17 3622 4.5 155.1 27.6 FIX-(CTP)₂ 10.449105.7 12 165.4 10.9 harvest rhFIX 3.72 1416.8 5.1 373.8 70.183 PurifiedFIX- 11.14 6314.2 12.3 254.5 15.83 CTP-CTP

The FIX-CTP-CTP harvest demonstrated an improved PK profile compared toFIX-CTP harvest. Furthermore, purified FIX-CTP-CTP exhibited a 3-foldincrease in T½_(β) value and a 4.5-fold increase in AUC compared torhFIX.

The reduced amount of secreted FIX fused to tandem CTP molecules versusfusion of a single CTP appears to be due to the addition of an extra CTPand not to reduced detection by ELISA, because the Bradford-purifiedFIX-CTP-CTP calculated concentration was similar to the ELISA-calculatedconcentration.

FIX-CTP-CTP clotting activity was similar to pooled human plasma;however, its in vitro chromogenic activity was significantly lower whencompared to rhFIX or pooled human plasma. The chromogenic activity assaywas reported as a very sensitive assay compared to the coagulationassay. The reason for reduced activity of FIX-CTP-CTP may vary. Additionof CTP may decrease the affinity of FIX to FXIa or reducepost-transcriptional modifications (e.g. 12-10 GLA residues andpro-peptide cleavage). N-terminal analysis revealed that the proteolyticcleavage of the FIX-CTP-CTP pro-peptide was not fully completed prior tosecretion. Since this post-transcriptional modification is crucial forthe normal enzymatic activity of the protein, co-transfection withFurine-PACE plasmid is favorable and may improve FIX-CTP-CTP activity.

Finally, FIX-CTP-CTP comparative PK study in rats demonstrated thatfusion of two tandem CTPs to the C-terminal of FIX generated a FIX withan extended half-life.

FIX Depleted Mouse Model:

In order to assess the in vivo activity, FIX knockout mice are obtained,and a breeding colony is established. 10 μg of either commercialrecombinant hFIX (BeneFIX®) or rFIX-(CTP)₂ (FIX-CTP-CTP) are injectedinto the tail vein of an anaesthetized FIX knockout mouse (22-28 g). Theamount of injected protein equals to the required concentration of FIXin normal plasma (5 μg/ml). Blood samples are taken from the clippedtail into heparinized capillary tubes at specific time points. Plasmasamples are assessed for FIX levels by ELISA and efficacy is measured byaPTT coagulation assay.

Increasing FIX Propeptide Cleavage Efficacy:

CTP peptide cDNA was fused to the 3′ end of human FIX cDNA. Thecorresponding rFIX and Furin expressing constructs were co-transfectedinto Dg44 cells; a human rFIX cDNA was also co-transfected with theFurin plasmid as a control. Secretion of high level of FIX leads tosecretion of a mixture of pro-factor and a mature factor FIX, due tolimited amount of the Furin protease in the cell. Co-transfection of aFurin expressing vector with a pro-factor expressing vector increasesthe recovery and result in the secretion of fully processed FIX in tothe medium.

Following FIX-(CTP)₂ and Furin co-transfection, stable clones aregenerated and harvest is collected for pro-peptide cleavage evaluation.100 ng of protein, are loaded on 12% Tris-Glycine gel using PrecisionPlus Dual Color Protein Marker (Bio-Rad). The SDS-PAGE analysis isperformed by Western immunoblot using anti-human FIX polyclonal Ab(American Diagnostics) and anti-pro-peptide polyclonal antibody. Aspreviously reported, rhFIX migrated at 55 KDa, while FIX fused to twoCTPs migrated at 75 kDa. Both variants of FIX proteins are shown toundergo a proper, full pro-peptide cleavage.

To determine whether proper pro-peptide cleavage improves FIX-(CTP)₂enzymatic activity, a comparative assessment of chromogenic andcoagulation activity of FIX-(CTP)₂ harvest cotransfecated with Furin isperformed. A significant improvement in FIX-(CTP)₂ specific activity isobserved, which is similar to rhFIX.

In conclusion, the results described herein suggest that FIX-CTP-CTP canbe used efficiently for treating Hemophilia B patients. FIX fused to CTPconstructs benefit from improved in vivo pharmacologic performance thatovercomes the drawback in certain in vitro measures. This proposedtreatment is advantageous over previous treatments as the rate ofinfusions and the amount of required doses are reduced.

It is important to notice that when an albumin-fused molecule strategywas used to improve the FIX half-life, the recombinant FIX becameinactive. The present novel approach lead to the design and purificationof a novel recombinant FIX-fused protein that presents an improvedlong-lasting activity. Since mere size modifications did not improve thepharmacokinetics of injected FIX, the finding that CTP fused to FIXfacilitates pharmacokinetic parameters was unexpected. The presence ofhighly glycosylated peptide-sialic acid residues stabilized the proteinand protected it from interactions with vascular receptors withoutabrogating key determinants of FIX function.

FIX-CTP has a similar therapeutic efficacy to rFIX in hemophilia Bpatients and required less frequent dosing. A single injection ofFIX-CTP is sufficient to control bleeding episodes and reduce the numberof injections that are needed during surgical intervention in hemophiliaB patients.

The CTP technology was utilized for the development of a long-actingFIX. Specifically, extending the half-life of recombinant rFIX moleculewas performed by fusion of at least one human CTP to FIX. Therecombinant FIX-CTP was expressed in mammalian cells and characterizedin vitro and in vivo. It was demonstrated that the in vitro activity ofrFIX-CTP was comparable to rFIX. Pharmacokinetics and efficacy studiesin rats demonstrated improved properties of the rFIX-CTP. The results ofthis study demonstrate that it is feasible to develop a half-lifeextended rFIX molecule having similar haemostatic properties to the wildtype enzyme.

Example 2 Comparative Assessment of Purified FIX-CTP₃ vs. FIX-CTP₄ andFIX-CTP₅ 2.1 Study Objective

A comparative assessment of the pharmacokinetic parameters of FIX-CTP₄and FIX-CTP₅ versus FIX-CTP₃ following a partial purification process.

2.2 Production of FIX-CTP₄ and FIX-CTP₅ Harvests

FIX cDNA (OriGene RC219065) fused at the C-terminal to four or fivetandem CTP sequences was expressed in Dg44 cells using Excellgeneexpression system in the presence of 10 ng/L of vitamin K3 (Sigma,Mennadion). The harvests were collected (300 ml), filtered and frozen.

2.3 Production of FIX-CTP₃ Harvest

FIX-CTP₃ was expressed in-house in CHO cells using pCI-DHFR vector,clone 196, BR-9 in the presence of 25 ng/L of vitamin K3 (Sigma). Theharvests were collected and filtered.

All FIX-CTP samples (3, 4 and 5 CTP) were purified only by Jacalincolumn because of a lack of material.

2.4 Determination of FIX Antigen Level

FIX antigen level was determined using Human FIX ELISA kit (AffinityBiologicals; Cat. #FIX-AG RUO). The calculated protein concentration isthe average of four independent runs. FIX-CTP₃ concentration wasslightly higher as compared to the two additional versions (Table 10).

TABLE 10 FIX antigen level 3 CTP 4 CTP 5 CTP Final Final Final Jacalin40Jacalin40 Jacalin40 Av. (ng/ml) 1016.69 4644.11 1686.82 SD 225.41 925.63160.07 % CV 22.17 19.93 9.49

2.5 FIX-CTP Coomassie Stain and Immune-Blot

FIX-CTP₃, FIX-CTP₄, and FIX-CTP₅ harvests were loaded on 12%Tris-Glycine gel using Precision Plus Dual Color Protein Marker(Bio-Rad). The SDS-PAGE analysis was performed by Western immuno-blotusing anti-CTP polyclonal Ab (Adar Biotech Production) or anti-Gla Ab(American Diagnostica).

As previously reported, FIX fused to three CTPs migrated at 80 kDa whileFIX fused to four or five CTPs migrated at 85 KDa or 90 KDa,respectively. As expected, FIX-CTP₄ and FIX-CTP₅ harvests fromExcellgene showed very low levels of gamma carboxylation compared toFIX-CTP₃ harvest, which was produced at Prolor (FIG. 8).

After a purification process utilizing Jacalin column (immunoaffinitypurification of glycosylated proteins), FIX-CTP₃, FIX-CTP₄, and FIX-CTP₅were loaded on 12% Tris-Glycine gel using Precision Plus Dual ColorProtein Marker (Bio-Rad). The SDS-PAGE was stained by Coomassie blue Dyefor samples detection. All variants showed much cleaner band profiles(FIG. 9), suggesting an improved purity.

2.6 Determination of FIX Chromogenic Activity

A comparative assessment of the in vitro potency of fully purified (HAcolumn) FIX-CTP₃, FIX-CTP₄, and FIX-CTP₅ versus human pool normal plasmawas performed using a commercially available chromogenic activity testkit, BIOPHEN (Hyphen BioMed 221802). All samples were serially diluted,and the potency was assessed by comparing a dose-response curve to areference preparation of normal human plasma. The reduced chromogenicactivity of FIX-CTP₄ and FIX-CTP₅ (FIG. 10) as compared to plasma can bea consequence of improper post-transcriptional modifications of FIXproteins, e.g. inappropriate gamma carboxylation and pro-peptidecleavage or, alternatively, due to the addition of CTP cassettes. Thefluctuation in the FIX-CTP₄ and FIX-CTP₅ activity (Table 11) might becaused by inappropriate quantitation capabilities of the FIX ELISA dueto CTP masking of the antigen site.

TABLE 11 Sample/plasma EC50 ratio Sample/plasma Sample EC50 ratio Plasma1 3 CTP Final HA 2 4 CTP Final HA 5.35 5 CTP Final HA 2.73

2.7 Pharmacokinetic Study

Jacalin-purified FIX-CTP₃, FIX-CTP₄, and FIX-CTP₅ (Group A, B and C,respectively) were administered in a single intravenous injection toSprague-Dawley rats (six rats per treatment group) at a dose of 250μg/kg body weight. Blood samples were drawn retro-orbitally from 3 ratsalternately at 0.083, 0.5 2, 5, 8, 24, 48, 72 and 96 hours post-dosing(Table 12). Citrated plasma (0.38%) was prepared immediately aftersampling and stored at −20° C. until analysis.

TABLE 12 PK study plan of operation No. of Dose Level Injected Treatmentanimals/ Dose (μg per Vol. Conc. Time-Points Group Treatment group Routeanimal) (μl) (μg/ml) (hr post-dose) A FIX-CTP*3 6 IV 50 200 250 0.083,0.5, 2, 5, 8, Jacalin 40 24, 48, 72, 96 B FIX-CTP*4 6 IV 50 200 2500.083, 0.5, 2, 5, 8, Jacalin 40 24, 48, 72, 96 C FIX-CTP*5 6 IV 50 200250 0.083, 0.5, 2, 5, 8, Jacalin 40 24, 48, 72, 96

FIX concentration in plasma samples were quantified using human FIXELISA kits (Affinity Biologicals). The pharmacokinetic profile wascalculated and is the mean of 3 animals at each time point. Terminalhalf-lives were calculated using PK Solutions 2.0 Software. Table 13below summarizes the calculated FIX concentrations at the differentsampling time points.

TABLE 13 Calculated FIX concentrations Av. 3 Av. 4 Av. 5 Time CTP SD 3CTP SD 4 CTP SD 5 (hr) ng/ml CTP ng/ml CTP ng/ml CTP 0.083 1087.82 72.39904.54 21.06 1097.23 82.24 0.5 774.18 86.31 736.82 66.93 998.79 70.43 2562.23 3.70 627.09 32.47 747.85 14.02 5 357.44 8.63 431.23 29.41 576.4927.36 8 239.20 7.82 327.46 30.26 394.96 36.48 24 77.08 4.26 107.38 5.18142.42 16.13 48 27.73 2.02 39.83 1.85 53.66 3.33 72 12.55 1.48 21.531.55 23.54 3.32 96 6.66 1.23 10.63 0.13 18.54 3.39

The PK profile and a summary of the PK parameters are presented in Table14 below and in FIG. 11. A full PK analysis profile at all time pointssuggested that addition of 4 or 5 CTP cassettes to FIX did not increaseits half-life as compared to FIX-CTP₃. The AUC following FIX-CTP₅administration increased by 1.4- to 1.6-fold versus FIX-CTP₃, which wasnot statistically significant.

TABLE 14 PK profile and a summary of the PK parameters 24-96 hr 3 CTP 4CTP 5 CTP Half-life (hr) 20.43 22.02 23.96 AUC (ng-hr/ml) 8218.3810504.49 13329.41 Vd (ml/kg) 700.76 586.02 494.89 CL (ml/hr/kg) 23.7718.45 14.32

Since 96 hr post-dosing samples were shown to have very low FIXconcentrations, which were at the lower limit of quantification of theassay, the terminal half-life was recalculated providing a more preciseand scientifically appropriate calculation (Table 15). According to thiscalculation, even smaller differences were obtained between thehalf-life of FIX-CTP₃, FIX-CTP₄, and FIX-CTP₅.

TABLE 15 Recalculated terminal half-life 8-72 hr 3 CTP 4 CTP 5 CTPHalf-life (hr) 15.38 16.63 16.04

2.8 Conclusions:

In this study, the pharmacokinetic parameters and potential clottingactivity of FIX-CTP₃, FIX-CTP₄, and FIX-CTP₅ were assessed. Fusion of 4and 5 CTPs to FIX did not provide a superior or improved half-lifeextension, as compared to FIX-CTP₃, and reduced chromogenic activity wasobserved. Table 16 below summarizes the percent improvement of half-lifefor the different FIX-CTP fused variants (1 to 5 CTPs). Fusion of CTP toFIX improved its pharmacokinetic behavior, but, unpredictably, thisimprovement was limited. Surprisingly, following fusion of 3, 4 or 5CTPs in tandem to FIX, a similar half-life value was calculated.

TABLE 16 Summary of the percent improvement of half-life T½ (8-72 hr)FIX Version % increase rhFIX vs. 1CTP 112 1CTP vs. 2CTP 141 2CTP vs.3CTP 37 3CTP vs. 4CTP 6 4CTP vs. 5CTP 0

These data suggest that fusion of 3 CTPs to FIX produces a maximalimprovement in protein half-life, confirming that FIX-CTP₃ is theoptimal variant in terms of half-life, structure and potential clottingactivity for further clinical development.

Example 3 FIX-CTP₃ Treatment of FIX−/− Hemophilic Mouse Model

As described above, a study testing FIX-CTP, FIX-CTP₂ and FIX-CTP₃harvest PK profile and coagulation activity vs. rhFIX was conducted.FIX-CTP₃ exhibited an improved PK profile while maintaining itscoagulation activity vs. FIX-CTP₁ and FIX-CTP₂ harvests or rhFIX. Tofurther evaluate this result, FIX-CTP₃ γ-Carboxyglutamate protein waspurified. FIX-CTP₃ exhibits a 3-fold increase in half-life and 4.5-foldhigher AUC compared to rhFIX in normal rats following a single IVadministration. FIX-CTP₃ demonstrated a reduced in vitro chromogenic andclotting activity, most likely due to insufficient cleavage ofN-terminal pro-peptide and in appropriate post-transcriptionalmodifications (PTMs), such as appropriate gamma carboxylation.

In the current study, the pharmacokinetic and pharmacodynamic propertiesof human recombinant FIX fused to three tandem CTPs were tested inFIX-deficient mice.

Study Purpose:

To determine the pharmacokinetic and pharmacodynamic parameters ofrFIX-(CTP)₃ vs. commercial rhFIX (BeneFIX®) in FIX-deficient micefollowing a single IV administration of FIX-(CTP)₃ at a similar specificactivity and dose (similar specific activity to PD and similar FIXconstant for PK).

Production of FIX-CTP₃ Harvest:

FIX cDNA (OriGene RC219065-Thr 148) fused at the C-terminal to threetandem CTP sequences was expressed in Dg44 cells using Excellgeneexpressing system in the presence of 25 ng/ml of Vitamin K3 (Sigma,Mennadion). Five separate batches containing 5 liters of cell suspensionwas cultured (total of twenty-five liters) and harvested followingviability decline to 60-70%. The harvest was filtered and frozen at −70°C.

Determination of Harvest FIX Antigen Level:

Harvest FIX antigen level was determined using a human FIX ELISA kit(Affinity Biologicals; Cat. # FIX-AG RUO). The antigen level wascalculated per each batch. The FIX concentration was maintained throughthe different batches (Table 17).

TABLE 17 FIX antigen level FIX antigen level Batch #1 Bat #2 Bat #3 Av(μg/ml) 28.81 32.74 42.9 STD 2.5 2.69 4.0 % CV 8.84 8.38.2 9.4

FIX-CTP₃ Purification Process:

Following a short purification study, a purification process using thefollowing 3 columns was performed: DEAE Sepharose, Heparin Sepharose andHA Bio Rad Ceramic Hydroxyapatite type 1 (40 μm), FIX-CTP₃.γ-carboxylated enriched protein was purified. In brief: Five liters ofclarified harvest was thawed at 4° C. over a 4 day period. For eachpurification batch, the clarified harvest (2 liters) was concentrated4-fold and dialyzed against 20 mM Tris-HCl pH 8.2 using a disposablehollow fiber cartridge with a nominal molecular weight cutoff size of 10KDa. This process (UFDF1) was performed twice, and one liter of UFDF1was loaded on DEAE Sepharose column, and Factor IX was eluted with 20 mMTris-HCl, 200 mM NaCl, 10 mM CaCl₂ pH 8.2. The product was diluted 1:1with 20 mM Tris-HCl, 10 mM CaCl₂ pH 7.5, and the pH was adjusted to 7.5before loading on Heparin Sepharose column. The elution was performedwith 20 mM Tris-HCl, 300 mM NaCl, and 10 mM CaCl₂ pH 7.5. The elutedproduct was concentrated and dialyzed against 10 mM phosphate pH 6.8using a Pellicon XL cassette 10 KDa cutoff membrane (UFDF2). The productwas loaded on an HA column, and the activated fraction of Factor IX waseluted with 150 mM phosphate pH 6.8. The purification product wasconcentrated to a target concentration of 2 mg/ml and dialyzed againstTBS pH 7.45, divided in aliquots and stored at −70° C.

The purification process was repeated five times, on a weekly basis inorder to purify the total volume (25 liters). The purification processeswere named HA#6-10. Each purification product was separately evaluated(App #1-5). At the end of the purification process, the differentbatches were pooled and further concentrated to a target concentrationof 4 mg/ml.

FIX-CTP₃ Analytical Properties:

Determination of FIX Antigen Level

FIX-CTP₃ γ-carboxylated enriched protein antigen level was determinedusing a human FIX ELISA kit (Affinity Biologicals; Cat. # FIX-AG RUO).The calculated protein concentration is the average of two independentruns (Table 18).

TABLE 18 FIX-CTP₃ antigen level FIX-CTP₃ HA purified pool ELISA #1FIX-CTP₃ HA purified pool- ELISA #2 Final Dil. 1 2 Av. Dil. 1 2 Av. Av.130000 3412240 3781830 3597035 130000 3692260 3568240 3630250 3613643260000 3915600 4158440 4037020 260000 3706820 3595540 3651180 3844100520000 4158544 4334096 4246320 520000 3831464 3530748 3681106 39637131040000 4096352 4004104 4050228 1040000 3863392 3684304 3773848 3912038Av. 3895684 4069618 3982651 Av. 3773484 3594708 3684096 3833373 (ng/ml)(ng/ml) STD 338367.5 234486.7 274313.5 STD 86576.66 65369.65 63369.86154459.6 % CV 8.685703 5.761884 6.887712 % CV 2.294343 1.818497 1.7200924.029338 Av. 3.895684 4.069618 3.982651 Av. 3.773484 3.594708 3.6840963.833373 (mg/ml) (mg/ml) 130000 3412240 3781830 3597035 130000 36922603568240 3630250 3613643 260000 3915600 4158440 4037020 260000 37068203595540 3651180 3844100 520000 4158544 4334096 4246320 520000 38314643530748 3681106 3963713 1040000 4096352 4004104 4050228 1040000 38633923684304 3773848 3912038 Av. 3895684 4069618 3982651 Av. 3773484 35947083684096 3833373 (ng/ml) (ng/ml) STD 338367.5 234486.7 274313.5 STD86576.66 65369.65 63369.86 154459.6 % CV 8.685703 5.761884 6.887712 % CV2.294343 1.818497 1.720092 4.029338 Av. 3.895684 4.069618 3.982651 Av.3.773484 3.594708 3.684096 3.833373 (mg/ml) (mg/ml)

SDS-PAGE Blots:

FIX-CTP₃ γ-carboxylated enriched protein, rhFIX and rFIXa (activatedFIX) were loaded on 12% Tris-Glycine gel using Precision Plus Dual ColorProtein Marker (Bio-Rad). The SDS-PAGE Coomassie analysis was performedby staining the gel with Coomassie blue reagent (800 ng of protein)(FIG. 12). A Western immunoblot was performed using 100 ng of proteinwith anti-human FIX polyclonal Ab (FIG. 12B), anti-human gammacarboxylation monoclonal antibody (American Diagnostics Cat #499, 3570)(FIG. 12C), anti-FIX pro-peptide polyclonal Ab (FIG. 12D), and anti-CTPpolyclonal Ab (FIG. 12E). As previously reported, FIX-CTP₃ migrated at75 KDa.

The purification procedure significantly enriched FIX-CTP₃ portion whilereducing impurities. The purification process yield was very low rangingaround 2-3% (data not shown) due to the requirement to collect only theγ-carboxylated FIX-CTP₃ fractions, as demonstrated in the anti-Glaimmunoblot (FIG. 12B). Based on the Coomassie and FIX immunoblot, theFIX-CTP₃ portion is only around 60-70%, and additional lower molecularweight bands, presumably with lower glycosylation forms, were alsodetected.

FIX-CTP₃ Clotting Activity:

FIX-CTP₃ Chromogenic Activity:

A comparative assessment of the in vitro potency of FIX-CTP₃ harvest andFIX-CTP₃ γ-carboxylated enriched protein, versus human pool normalplasma was performed using a commercially available chromogenic activitytest kit, BIOPHEN (Hyphen BioMed 221802). FIX-CTP₃ harvest and proteinwere serially diluted, and the potency was assessed by comparing adose-response curve to a reference preparation consisting of normalhuman plasma. As previously demonstrated, FIX-CTP₃ harvest was 50 timesless active then human pool plasma (Table 19, FIG. 13). FollowingFIX-CTP₃ purification, the chromogenic activity was significantlyimproved and was only 4.72 times less active then human pool plasma(Table 19, FIG. 13). Harvest reduced chromogenic activity can be aconsequence of improper post-transcriptional modifications of FIXprotein variants, e.g. inappropriate gamma carboxylation and pro-peptidecleavage. Following purification and enrichment of the FIX-CTP₃γ-carboxylated fraction, the activity was improved, demonstrating theimportant contribution of γ-carboxylation to FIX activity.

TABLE 19 FIX-CTP₃ chromogenic activity EC₅₀ Sample/plasma Sample (ng/ml)EC₅₀ ratio FIX-CTP₃ 741.3 54.4 Harvest Pur. 64.6 4.72 FIX-CTP₃ Plasma13.63 1

One Stage Clotting Assay (aPTT):

The activated partial thromboplastin time (aPTT) is a measure of theintegrity of the intrinsic and common pathways of the coagulationcascade. The aPTT is the time, in seconds, for plasma to clot followingthe addition of an intrinsic pathway activator, phospholipid andcalcium. The principal of the assay was to quantitate the ability ofFIX-CTP₃ to restore the clotting activity of FIX-depleted human plasmaby the addition of rhFIX. 200 μl of FIX-deficient human plasma was mixedwith 25 μg/ml of FIX-CTP₃ and further diluted in TBS. Following a 60second incubation at 37° C., 50 μl of PTT activator (Actin FS) and 50 μlof calcium 25 mM were added to the mixture, and the clotting time inseconds was determined using a Sysmex® CA 1500 Coagulator (performed bySheba hospital, National Coagulation Center using validated aPTT assay).The potency was assessed by comparison of FIX-CTP₃ to the dose-responsecurve of a reference preparation of normal human pool plasma. Theresults are expressed in percent of activity interpolated from thestandard curve covering FIX levels of <1-110%. FIX-CTP₃ exhibited a15-20-fold reduction in its coagulation activity versus normal humanpool plasma since the activity at 5 μg/ml, which is the normal value ofFIX in the body, was shown to be 6.5% (Table 20).

TABLE 20 FIX-CTP₃ clotting activity FIX FIX % of activity ConcentrationConcentration in (normalized to by provider tested sample human normal(mg/ml) (μg/ml) pool plasma) Fix-CTP₃ 3.83 25 34.7 5 6.5

FIX-CTP₃ also exhibited increased clotting time compared to BeneFIX®(Table 21 and FIG. 14).

TABLE 21 Comparative clotting time (aPTT) Clotting time FIX-CTP₃BeneFIX ® 38 ug/ml 77.6 19 ug/ml 83.4 7.6 ug/ml 93.2 50.6 3.8 ug/ml104.8 57.6 1.9 ug/ml 112.2 63.7 0.95 ug/ml 122.6 71.5 0.475 ug/ml 83.70.238 ug/ml 94.3

An additional clotting assay was performed independently inFIX-deficient mice by Dr. Paul Monahan at University of North Carolinaprior to the initiation of the PK-PD study. The aPTT results suggestedthat FIX-CTP₃ coagulation activity is 40 times less than normal pooledhuman plasma as demonstrated by the longer period (as measured inseconds) and higher concentration that are required for proper clottingactivity (Table 22).

TABLE 22 Comparative clotting activity FIX activity (Units) FIX-CTP₃BeneFIX ® 38 ug/ml 13.9 19 ug/ml 8.8 7.6 ug/ml 4 116.8 3.8 ug/ml 1.667.4 1.9 ug/ml 0.9 41.7 0.95 ug/ml 0.4 22.4 0.475 ug/ml 8.5 0.238 ug/ml3.7

The specific activity (u/ml), which was based on FIX antigen level ascalculated by ELISA for FIX-CTP₃ and BeneFIX®, was 4.46 and 198.9respectively.

The inconsistency in the calculated FIX-CTP₃ activity as demonstrated inthe chromogenic vs. aPTT assays can be explained by the superiorsensitivity of the aPTT assay and in vivo relevance. In the chromogenicactivity assay, an excess amount of reagents and enzymes are presentwhich can activate less potent FIX versions. The difference in theFIX-CTP specific activity values can be explained by the use ofdifferent reagents and automated machines. The activity value ascalculated at University of North Carolina was used for the PK-PD studydesign.

FIXa Protein Detection:

In order to confirm that following the purification process, FIXactivation (FIXa) did not occur, a FIXa detection assay was performedusing FIXa Biophen Chromogenic Assay (Cat. # Ref. 221812). The assaymeasures the amount of FIXa present in a specific sample using thechromogenic activity cascade, as previously described. FIX-CTP₃ andrhFIX were diluted and FIXa levels were evaluated. FIX-CTP₃ wasn'tactivated through purification or storage (Table 23).

TABLE 23 FIXa detection Sample FIX-CTP₃ rhFIX Initial Con.(mg/ml) 10005.7 rFIXa (mg/ml) BLQ 0.00487 % FIXa in sample BLQ 0.085

FIX-CTP₃ PK-PD Study:

FIX-CTP₃ and rhFIX (BeneFIX®) were administered in a single intravenousinjection to C57BI FIX-deficient mice in a dose of 625 μg/kg body weightcontaining 100 IU FIX/kg body weight. Blood samples were drawnretro-orbitally from 3 mice alternately at 0.25, 4, 24, 48, 72, and 96hours post-dosing. Citrated plasma (0.32%) was prepared immediatelyafter sampling and stored at −20IC until analysis. hFIX antigen levelwas evaluated, and a detailed PK analysis was performed. In order toevaluate the ability of FIX-CTP₃ to elongate the clotting activity ofFIX-deficient animals compared to BeneFIX®, FIX activity in citratedplasma samples, collected from the FIX−/− treated mice, was calculatedusing an automated FIX activity assay (Table 24).

TABLE 24 Study outline # Collection Points Required ProductAdministration Dose mice (hr post-dosing) amount **Cohort 1 FIX-CTP₃Single 100 IU/Kg 12 0.25, 1, 4, 8, 16, 6636 μg dose: IV 2.5 IU/mousemice, 24, 48 (553 μg/mouse) Cohort 2 FIX-CTP₃ Single **472 μg/Kg 18*0.25, 1*, 4*, 8*, 200 μg dose: IV 12.57 μg/mouse mice 16*, 24*, 48*,72*, 12.57 μg/mouse 96* **Cohort 3 BeneFIX ® Single 100 IU/Kg 18 0.25,1, 4, 8, 16, 226.3 μg dose: IV 2.5 IU/mouse mice, 24, 48, *72, *96 12.57μg/mouse *PK collection points only **Tail vein bleeding at T = 48post-dosing; cohorts 1 & 3

FIX-CTP₃ Pharmacokinetic Profile in FIX^(−/−) Mice

FIX concentration was quantitated using human FIX ELISA kits (AffinityBiologicals; Cat. # FIX-AG RUO). The pharmacokinetic profile wascalculated for each protein and is the mean of three animals at eachtime point. Table 25 below and FIG. 15 summarize the calculated FIXconcentrations at the different sampling time points for Cohorts 1 & 3.The PK profile and a summary of the PK parameters are presented below(Tables 26 & 27). A PK analysis was also performed for Cohort #2 inorder to verify exposure (data not shown).

TABLE 25 FIX concentrations Time FIX-CTP₃ BeneFIX ® point(hr) ng/mlng/ml 0.25 3645.397 2823.023 1 2411.09 2416.248 4 1703.205 1506.228 81139.736 864.764 16 415.32 347.465 24 238.37 158.7973 36 141.010594.40067 48 95.461 42.28833 72 76.90953 11.87567 96 24.955 BLQ

A two-compartmental module was used (WinLin software) to determineAUC0-inf, T_(terminal) and clearance (CL). The PK parameters aredescribed below in Table 26.

TABLE 26 PK properties FIX T½α T½ β AUC CL MRT Vss Version (1/hr) (1/hr)ng/ml*hr ml/Kg/hr (hr) (ml/Kg) BeneFIX ® 3.4 12.7 22428 29 11.5 320.8FIX-CTP₃ 4 28.7 31770 19 22 425.2

The addition of the three CTP “cassettes” to rhFIX elongated FIXhalf-life in vivo by at least 2.5-fold. AUC following in vivo FIX-CTP₃administration increased 2-fold versus rhFIX. FIX-CTP₃-injected micedemonstrated an improved PK profile compared to BeneFIX®-injected mice.

FIX-CTP₃ Pharmacodynamic Profile in FIX-deficient Mice:

In parallel to PK sampling, FIX-deficient animals administered witheitherp BeneFIX® or FIX-CTP₃, citrated plasma samples, were evaluatedfor their clotting activity by aPTT assay, which was translated to %activity. The % activity at each collection point was calculated as thecurrent clotting time/clotting time of normal pool mice plasma* 100.Table 27 summarizes the activity values following administration ofeither BeneFIX® or FIX-CTP₃.

Following FIX-CTP₃ administration, significant clotting activity wasdetected one hour after administration reaching 96% activity at fourhours post-dosing, while BeneFIX® highest activity value was 40% (Table27, FIG. 16). FIX-CTP₃ clotting activity was maintained for a longerperiod of time, demonstrating elongated activity. Clotting activity forthe BeneFIX®-treated mice was undetectable at time points later than 36hours, while FIX-CTP₃-treated mice continued to retain measurableactivity at 72 hours post-dosing (Table 27, FIG. 16). Analysis of the %clotting pharmacokinetic profile suggest that FIX-CTP₃ clotting activityis maintained for a significantly longer period and its half-life isalmost 2-fold higher than Benefix® (Table 28).

TABLE 27 FIX % of activity BeneFIX ® FIX-CTP₃ Hr post-dosing % ofactivity % of activity 0.25 39.9 1.0 1 33.4 15.5 4 24.9 93.6 8 18.8 65.216 10.3 39.9 24 1.7 11.9 36 1.4 11.0 48 <1 4.6 72 <1 1.4

TABLE 28 Clotting Activity FIX T½α T½ β Version (1/hr) (1/hr) BeneFIX ®5.7 — FIX-CTP₃ 7.3 16

9.3 FIX-Deficient Mice Bleeding Challenge

FIX-deficient mice were administered a single intravenous injection of100 IU/kg of BeneFIX® or rFIX-CTP₃. The tail vein was slightly clipped48 hours post-dosing, and tail vein bleeding time (TVBT) and bleedingintensity (hemoglobin OD) were evaluated. A second bleeding challengewas performed 15 minutes after reaching homeostasis, and the sameparameters were measured. Following the first bleeding challenge,FIX-CTP₃-administered animals' bleeding was significantly less intensethen BeneFIX® bleeding as demonstrated by the Hemoglobin OD values (FIG.17).

Since it was previously reported that during the first bleedingchallenge in hemophilic mice, the bleeding time does not necessarilycorrelate with treatment efficacy, it is recommended to evaluate thehomeostasis following additional bleeding. Once the first bleeding wasspontaneously or manually stopped, a second bleeding challenge wasperformed 15 minutes following the first one, and the time and bleedingintensity were re-measured. During the second bleeding episodeFIX-CTP₃-administered animals had reduced bleeding time and intensity,demonstrating that FIX-CTP₃ was potent at a later time points (FIG. 18).

Finally, the animals were further observed for the 12 hours followingthe second bleeding challenge, and all recurring bleeding events weredocumented. FIX-CTP₃-administered animals were able to maintain bloodhomeostasis for the next 12 hours with no re-occurring bleeding events.In contrast, 50% of BeneFIX®-treated mice had spontaneous bleedingepisodes from the tail (Table 29).

TABLE 29 Outcome 12 hours after tail transection Delayed Death orDistress Mouse group rebleeding Requiring Euthanasia FIX-CTP₃ 0/5 (0%)0/5 (100 IU/kg) BeneFIX ® 3/6 (50%) 0/6 (100 IU/kg) FIX−/− (untreated)5/6 (100%) 1/6

Recombinant FIX-CTP₃, a fusion protein comprised of a single molecule ofFIX fused to three CTP “cassettes” in tandem was developed to addressthe short half-life of currently available FIX products used to treatpatients with hemophilia B. The results herein demonstrated that theelimination half-life of rFIX-CTP₃ was consistently 2.5- to 4-foldlonger than rFIX in rats (as previously reported) and in FIX-deficientmice.

Without being bound by theory, the fusion protein reduces clearance ofFIX and protects FIX from protease activity, degradation by masking andreduces the affinity of FIX for hepatic receptors. Taken together thesecharacteristics of the CTP domain extend the half-life of FIX.

In addition to pharmacokinetic analysis of rFIX-CTP₃, we examined thepharmacodynamic properties of FIX-CTP₃ in FIX-deficient mice. rFIX-CTP₃and rFIX, were administered at comparable doses (in units) to compensatefor the clotting deficiency levels in FIX-deficient mice. However, theeffect of rFIX-CTP₃ in FIX-deficient mice was significantly prolonged toat least 76 hr after dosing, reaching a higher activity peak. FIX-CTP₃clotting activity began after a 1-hour delay compared to BeneFIX®. FIXactivation may be required since the addition of three tandem CTPs mighttheoretically mask the activation site and delay cascade onset.Following FIX-CTP₃ administration, a 100% peak activity was observed,while BeneFIX® activity was only 40%. The superior initial activity is avery important parameter and demonstrates that addition of 3 CTPs hasthe potential to improve recovery.

Prophylactic FIX replacement therapy for patients with hemophilia B aimsto maintain plasma levels of 1-2% normal clotting activity. The tailvein bleeding assay is a sensitive in vivo test that measures theability to maintain bleeding homeostasis at low activity valuesmimicking human bleeding homeostasis model. In response to tail veinbleeding challenge 48 hours post-dosing, rFIX-CTP₃-administered animalsmaintained blood homeostasis with shorter and less severe bleedingepisodes, demonstrating sustained clotting activity.

FIX is a complex protein that contains a number of functional domainswhich undergo extensive post-translational modifications. One of theessential post-translational modifications for FIX activity isgamma-carboxylation of the first 12 glutamic acids in the Gla domain byvitamin K-dependent γ-glutamyl carboxylase. This modificationfacilitates the binding of FIX to phospholipid membranes and, thus, iscritical to its function. FIX that is not gamma-carboxylated is notfunctional, and hence gamma-carboxylation is a rate-limiting step.

This PK-PD study was conducted using transiently transfected cells. Anextensive analytical evaluation of post-translational modifications isperformed on the stable FIX-CTP₃ protein produced and secreted fromstable optimized clone.

Based on the presented data, FIX-CTP₃ coagulation factor has thepotential to reduce the frequency of injections in patients receivingroutine prophylactic doses of FIX replacement therapy. It is anticipatedthat rFIX-CTP₃ can confer prolonged protection from bleeding followingeach dose of factor, decrease the overall units of factor needed totreat bleeding episodes, and/or maintain adequate hemostasis duringsurgical procedures with fewer injections.

Example 4 Generation and Utilization of Coagulation Factor FVII

A long-acting version of activated Factor VII (FVIIa) coagulation factorwill be useful for the treatment of patients with hemophilia A and B.FVIIa-CTP₃ recombinant protein has the clinical potential to improve thetreatment of hemophilia patients by reducing the frequency of infusionsand even by reducing the drug load, enabling a prophylactic treatmentapproach which can significantly improves a patient's quality of life,avoid spontaneous bleeding episodes and accumulated damage to the jointand other organs.

The generation of a recombinant FVIIa-CTP molecule with an extendedhalf-life based on fusion of FVII to a human CTP is described herein.The recombinant FVIIa-CTP was expressed in mammalian cells andcharacterized in vitro and in vivo. It was demonstrated that rFVII-CTPactivity was comparable to rFVII. Pharmacokinetic and efficacy studiesin rats demonstrated improved properties of rFVII-CTP. The results ofthis study demonstrated that it is feasible to develop a half-lifeextended rFVIIa molecule with very similar haemostatic properties to thewild-type enzyme.

Cloning and Expression of Recombinant FVII Molecule:

Several Factor VII clones were constructed in our eukaryotic expressionvector (pCI-dhfrr) (FIG. 19). Human MGC verified FL cDNA clone (IRCM)containing the sequence of homo sapiens coagulation Factor VII wasordered from “Open Biosystems” (OB-MHS4426). The following primers weresynthesized by Sigma-Genosys in the following sequence: Primer 67:5′CTCGAGGACATGGTCTCCCAGGCCC3′ (contains the 5′ end of Factor VII DNA andthe restriction site of XhoI) (SEQ ID NO: 5); Primer 68^(R): 5′TCTAGAATAGGTATTTTTCCACATG3′ (contains the restriction site of XbaI) (SEQID NO: 6); Primer 69: 5′ TCTAGAAAAAAGAAATGCCAGC3′ (contains therestriction site of XbaI) (SEQ ID NO: 7); and Primer 70^(R):5′GCGGCCGCATCCTCAGGGAAATGGGGCTCGCA3′ (contains the 3′ end of Factor VIIDNA and the restriction site of NotI) (SEQ ID NO: 8).

Cloning was performed in two sets of PCR reactions. The first reactionwas conducted with primer 67 and primer 68^(R) using a cDNA plasmid withthe Factor VII sequence (OB-MHS4426) as a template; as a result of thePCR amplification, a ˜534 bp product was formed, isolated and ligatedinto a TA cloning vector (Invitrogen, Catalog No: K2000-01). A XhoI-XbaIfragment containing the amino terminus of the Factor VII sequence wasisolated. The second reaction was conducted with primer 69 and primer70^(R) and again, a cDNA plasmid with the Factor VII sequence(OB-MHS4426) was used as a template. As a result of the PCRamplification, a ˜813 bp product was formed and ligated into TA cloningvector (Invitrogen, Catalog No: K2000-01). A XbaI-NotI fragmentcontaining the carboxy terminus of Factor VII sequence was isolated. Thetwo fragments were inserted into our eukaryotic expression vectorpCI-dhfr (triple ligation) to yield the 501-O-p136-1 clone.

Plasmid 501-p136-1 (Factor VII in pCI-dhfr vector) was digested withrestriction enzymes XhoI and KpnI. A fragment of ˜1186 bp was isolated.A partial Factor VII clone (1180 bp-1322 bp) followed by a CTP sequence,termination sequence and NotI sequence that was synthesized by GeneArt(0721543) was digested with restriction enzymes KpnI and NotI. Afragment of ˜253 bp was isolated. The two fragments were inserted intoour eukaryotic expression vector pCI-dhfr (triple ligation) to yield the501-1-p137-2 clone. pCI-dhfr-701-2-p24-2 was digested with restrictionenzymes XhoI and ApaI, and the large fragment (vector) was isolated.

pCI-dhfr-501-2-p137-2 (Factor VII-ctp ×1) was digested with restrictionenzymes XhoI and ApaI, and a ˜1200 bp insert was isolated. The vectorand insert were ligated to yield 501-2-p139-2. Dg44 cells were plated in100 mm tissue culture dishes and grown to confluence of 50-60%. A totalof 2 μg of DNA was used for transfection of one 100 mm plate using theFuGene reagent (Roche) in protein-free medium (Invitrogen CD Dg44). Themedium was removed 48 hours post-transfection and replaced with aprotein-free medium (Invitrogen CD Dg44) without nucleosides. After 14days, the transfected cell population was transferred into T25 tissueculture flasks, and the selection was continued for 10-14 days until thecells began to grow well as a stable clone. High-expressing clones wereselected and approximately 2×10⁷ cells were used to inoculate 300 ml ofgrowth medium in a 1700 cm² roller bottle (Corning, Corning N.Y.)supplemented with 5 ng/ml of Vitamin K3 (menadione sodium bisulfate;Sigma). The production medium (harvest) was collected after a rapiddecrease in the cell viability to around 70%. The production medium wasfirst clarified and then concentrated approximately 20-fold and dialyzedto PBS using flow filtration cassette (10 KDaMWCO; Millipore Corp,Billerica, Mass.).

Determination of FVII Antigen Level

The cDNA coding the CTP peptide was fused to the 3′ end of the cDNAcoding human FVII. The corresponding rFVII construct was transfectedinto Dg44 cells. As a control, a human rFVII cDNA was utilized. Theproduction medium (harvest) was collected, concentrated and the secretedrecombinant FVII was further evaluated. rFVII, rFVII-CTP andrFVII-CTP-CTP antigen levels were determined by AssayMax Human FVIIELISA kit (AssayPro) (FIG. 20A). There was no significant difference inthe secretion level of rFVII-CTP and rFVII-(CTP)₂ compared to nativerFVII.

SDS-PAGE Blots

SDS-PAGE analysis was done by loading 50 ng of either harvest, purifiedor activated rFVII protein. Samples were loaded on 12% Tris-Glycine gelusing Precision Plus Dual Color Protein Marker (Bio-Rad). The SDS-PAGEanalysis was done by performing a Western immunoblot using an anti-humanFVII monoclonal antibody (Ab) (R&D systems) or anti-CTP polyclonalantibody generated in Rabbit.

The level of rFVII antigen correlated with the detected protein level ina SDS-PAGE immunoblotted with anti-FVII Ab. rFVII-CTP migrated as asingle band, while the corresponding molecular weight of the FVIIcontrol was approximately 52 KDa (data not shown). Both proteins reactedwith antibodies specific for FVII on immunoblots. The rFVII-CTP alsoreacted with antibodies specific for CTP. rFVII was secreted in itszymogene form with no trace of activated protein.

FVII Chromogenic Activity:

rFVII, rFVII-CTP and rFVII-(CTP)₂ harvest activities were determinedusing a commercially available chromogenic test kit (AssaySense HumanFVII Chromogenic Activity Assay Kit (AssayPro). For functionalcharacterization of the rFVII-CTP and its ability to be furtheractivated (FVIIa), concentrated rFVII-CTP (harvests) were placed in acommercially available chromogenic test kit that measure the ability ofTF/FVIIa to activate Factor X to Factor Xa that in the presence of FXaspecific substrate releases a quantitated signal (AssayPro). Theaddition of the CTP peptide at the C-terminal of the rFVII protein didnot impair the FVII serine protease activity (FIG. 20B, 20C).

FVII Clotting Activity:

Prothrombin time (PT) measures the extrinsic pathway of coagulation. ThePT is the time (measured in seconds) it takes plasma to clot followingthe addition of an extrinsic pathway activator, phospholipid andcalcium. It is used to determine the clotting tendency of blood,specifically in the measure of warfarin dosage, liver damage, andvitamin K status. The reference range for prothrombin time is usuallyaround 12-15 seconds. Specifically, the assay quantitated the ability ofFVII-CTP and FVII-(CTP)₂ harvest to restore the clotting activity ofFVII-depleted human plasma by the addition of rhFVII. 300 μl ofFVII-deficient human plasma was mixed with 100 μl of FVII, FVII-CTP andFVII-(CTP)₂ harvests at specific concentrations, or normal pool humanplasma and were further diluted. Following a 60 second incubation at 37°C., Tissue Factor (TF), CaCl₂, and phospholipids were added to themixture. The clotting time in seconds was determined. Potency wasassessed by comparing a dose-response curve of FVII-CTP and FVII-(CTP)₂harvests to a reference preparation consisting of rhFVII or human poolplasma. One unit of active FVII was defined as the amount of FVII whichequals to the activity of one ml human normal plasma. The PT Clottingactivity of rFVII and rFVII-CTP was measured on a coagulometer(Instrumentation Laboratory).

As previously shown, the addition of a CTP peptide at the C-terminal ofthe rFVII protein did not damage its serine protease activity and leadto the initiation and activation of a native Factor X and Factor IX inhuman plasma. Following the insertion of an additional CTP at the Cterminal, there was a three-fold reduction in the serine proteaseactivity (data not shown).

Pharmacokinetics Study:

rFVII, rFVII-CTP, and rFVII-(CTP)₂ harvests were administeredintravenously to Sprague-Dawley rats (six rats per substance) with adose of 100 μg/kg body weight. For all of the in vivo experiments, theamount of the respective protein was determined on the basis of FVIIELISA kit. For each FVII test substance, the injected amount wascalculated by taking into account the differences in the molecularweight of rFVII versus rFVII-CTP, which leads to a different molarconcentration.

Blood samples were drawn retro-orbitally using an altering samplingscheme to minimize interference of the sampling procedure levels to bequantified: from 3 rats at 30 and 90 min and at 2, 6, and 48 hrs, andfrom the remaining three rats at 15 and 60 min and at 1.5, 4, and 24 hrsalternately. Plasma was prepared immediately after sampling and storedat −20° C. until analysis. FVII concentration was quantified by FVIIELISA specific assay. Half-life and area under the curve (AUC) werecalculated using a linear trapezoidal rule. Comparison of theseclearance parameters revealed that the in vivo half-life andrFVII-(CTP)₂ AUC are significantly higher than those of rFVII (Table30).

TABLE 30 PK study parameters Dose T½ AUC_(0-t) CL/F MRT Group Routeμg/kg min ng/min/mL mL/min/kg min FVII IV 60 4.07 3314.7 6.195 6.2 FVII-IV 60 β = 51.06 31353.9 0.287 73.7 CTP FVII- IV 60 β = 13.66 7626.8 1.1815.4 CTP-CTP

Characterization of Recombinant FVIIa-CTP:

During activation, FVII is cleaved at R152 resulting in heavy and lightchain domains that are held together by a single disulfide bridge.rFVIIa-(CTP)₂ is purified and activated by an ion exchange columnpurification process. In order to fully evaluate rFVIIa-(CTP)₂, theprotein is loaded on SDS-PAGE under reducing conditions to commercialFVIIa (NovoSeven®). The heavy and the light chain domains are separatedand migrate as separated bands of molecular weights 55 and 25 KDa. Bothproteins react with antibodies specific for FVII, but the heavy chain ofthe rFVIIa-CTP specifically reacts with anti-CTP-specific antibodies,indicating that this band represents the FVII heavy chain fused to CTP.The light chain reacts specifically with anti-gamma carboxylase Ab. TheFVIIa protein concentration is determined by FVIIa-specific ELISA kit.

FVIIa N-terminal Sequencing:

rFVII-CTP-CTP in activated or zymogene purified proteins is separated bySDS-PAGE (on 12% Tris-Glycine) and subsequently electroblotted to a PVDFmembrane. The bands of interest are cut out and put on a purifiedBiobrene-treated glass fiber filter. The N-terminal sequence analysis iscarried out by Edmann degradation using a pulsed liquid proteinsequencer equipped with a 140 C HPLC microgradient system. The identityof the recombinant protein and proper pro-peptide cleavage is furtherverified by N-terminal sequencing.

FVIIa Clotting Activity:

In order to evaluate FVII-(CTP)₂ coagulation activity, activated partialthromboplastin time assay (aPTT) is performed. FVII-deficient plasmasample is substituted with rFVIIa (NovoSeven®) or rFVIIa-(CTP)₂. 300 μlof FVII deficient human plasma is mixed with 100 μl of FVIIa orrFVIIa-(CTP)₂ at specific concentrations, or normal pooled human plasmawhich is further diluted. Following 60 seconds incubation at 37° C.Tissue Factor (TF), CaCl₂, and phospholipids are added to the mixture.Clotting time in seconds is determined. Potency is assessed by comparinga dose-response curve of rFVIIa-(CTP)₂ to a reference preparationconsisting of rhFVIIa or human pool normal plasma. One unit of FVIIa isdefined as the amount of FVIIa which equals to the activity of 1 mlhuman normal plasma. The aPTT clotting activity of rFVII andrFVIIa-(CTP)₂ is measured on a coagulometer (InstrumentationLaboratory). The aPTT clotting activity of rFVIIa and rFVIIa-(CTP)₂ issimilar.

Pharmacokinetics Studies in Rats:

In order to characterize the influence of the CTP addition to the rFVIIaon its longevity potential, a comparative pharmacokinetic study in ratsis performed. NovoSeven® (rFVIIa) and rFVIIa-(CTP)₂ in TBS are injectedIV to 6 SD rats. The levels of FVIIa over time are detected using aFVIIa ELISA kit. The half-life and AUC are calculated for each protein.Comparison of these clearance parameters reveals that the in vivomeasures of half-life, recovery, and AUC of the rFVIIa-(CTP)₂ aresuperior to those of NovoSeven®.

FVIIa-CTP in vivo Efficacy Model (FVIII-deficient Mouse Model ofHemophilia):

In order to assess the in vivo activity model, FVIII knockout mice areobtained, and a breeding colony is established. 10 μg of eithercommercial recombinant hFVIIa (NovoSeven®) or rFVIIa-(CTP)₂ are injectedinto the tail vein of an anaesthetized FVIII knockout mouse (22-28 g).The amount of injected protein equals to the required concentration ofFVIII in normal plasma (5 μg/ml). Blood samples are taken from theclipped tail into heparinized capillary tubes at specific time points.Plasma samples are assessed for FVIIa levels by ELISA, and efficacy ismeasured by a PTT coagulation assay.

In this study, a fusion construct of FVII with CTP is generated. Thisrecombinant protein is the basis for a treatment that provides aprolonged half-life and retention of therapeutic potency.

These results suggest that rFVIIa-(CTP)₂ has a similar therapeuticefficacy to rFVIIa in hemophilia patients. Moreover, this technologyrequires less frequent dosing. It appears that a single injection ofrFVIIa-(CTP)₂ is sufficient to control bleeding episodes and reduce thenumber of injections that are needed during surgical intervention. Thisrecombinant protein may be used as a long term prophylactic treatment.

Example 5 Comparative Assessment of Purified FVII-CTP₃, FVII-CTP₄, andFVII-CTP₅ 5.1 Study Objective

Comparative assessment of pharmacokinetic parameters and clottingactivity of FVII-CTP₄ and FVII-CTP₅ versus FVII-CTP₃.

5.2 Production of FVII-CTP₄ and FVII-CTP₅ Harvests

FVII cDNA fused at the C-terminal to four or five tandem CTP sequenceswas expressed in Dg44 cells using the Excellgene expressing system inthe presence of 20 μg/L of vitamin K3 (Sigma, Mennadion). The harvestwas collected (300 ml), filtered and frozen.

5.3 Production of FVII-CTP₃ Harvest

FVII-CTP₃ was expressed in-house in mammalian expressing system, CHOcells, using pCI-DHFR vector. Stable transfected pool #71 was grown inshake flasks, in the presence of 25 ng/L of vitamin K3 (Sigma). Theharvests were collected and filtered.

All FVII-CTP harvests (3, 4 and 5 CTPs) were concentrated and dialyzedagainst TBS (50 mM Tris, 150 mM NaCl, pH 7.4) using Pellicon XL MWCO 10kDa.

5.4 Determination of FVII Antigen Level

FVII antigen level was determined using Human FVII ELISA kit (ZymotestHyPhen) (Table 31). The calculated protein concentration is the averageof two independent runs.

TABLE 31 FVII antigen level FVII- FVII- FVII- CTP₃ CTP₄ CTP₅ Av. (ng/ml)224357.3 87884.1 589423 SD 44789.5 3248.7 5309 % CV 20.0 3.7 9

5.5 FVII-CTP Immune-Blot

FVII-CTP₃, FVII-CTP₄, and FVII-CTP₅ harvests were loaded on 12%Tris-Glycine gel (expedeon) using Precision plus dual color proteinmarker (Bio-Rad). The SDS-PAGE analysis was performed by Westernimmune-blot using anti-CTP polyclonal Ab (Adar Biotech Production) oranti-Gla Ab (American Diagnostica).

FVII fused to three, four and five CTP migrated at 80, 90 and 100 kDa,respectively. As expected, FVII-CTP₄ and FVII-CTP₅ harvests fromExcellgene contain low gamma carboxylation content as compared toFVII-CTP₃ harvest which was produced at Prolor since the productionprocess wasn't optimized (FIG. 21).

5.6 Comparative Assessment of FVII In Vitro Potency

A comparative assessment of the in vitro potency of HA purified (highlygamma carboxylated fraction) FVII-CTP₃, FVII-CTP₄, and FVII-CTP₅ versusnormal human pool plasma was performed using a commercially availablechromogenic activity test kit, BIOPHEN (Hyphen BioMed 221304). Allsamples were serially diluted, and the potency was assessed by comparinga dose-response curve to a reference preparation consisting of normalhuman plasma. FVII-CTP₃ and FVII-CTP₅ demonstrated chromogenic activitylower than pooled normal plasma (FIG. 22). FVII-CTP₄ demonstrated higheractivity as reflected by EC50 ratios, compared to FVII-CTP₃ andFVII-CTP₅ (Table 32).

TABLE 32 FVII In Vitro Clotting Activity Sample/plasma Sample EC50(ng/ml) EC50 ratio Plasma 0.05 FVII 3CTP 0.12 2.72 FVII 4CTP 0.03 0.71FVII 5CTP 0.06 1.35

5.7 FVII In Vitro Clotting Activity:

Factor VII (FVII) activity assay, which was performed in Sheba MedicalCenter, the Israel National Coagulation Center, is a prothrombin(PT)-based assay using immuno-adsorbed plasma deficient in Factor VII(Siemens). The PT reagent is innovin, and the assay is performed in theSysmex® CA 1500 instrument. FVII normal range is within 55-145%.

TABLE 33 FVII In Vitro Chromogenic Activity Concentration in testedsample Concentration Sample FVII % of activity (μg/ml) (μg/ml) FVII 3CTP36 0.5 224.2 18 0.25 6 0.125 FVII 4 CTP 334 0.5 87.9 176 0.25 93 6.25FVII 5 CTP 38 0.5 58.9 19 0.25 10 0.125

Since the normal level of circulating FVII in the body is around 0.5μg/ml, FVII-CTP₃ and FVII-CTP₅ harvests exhibit 3-fold reductions intheir coagulation activity versus normal human pool plasma; this resultcorrelates with the obtained chromogenic activity (Table 33).

The FVII-CTP₄ harvest exhibits a 3-fold increase in its potentialcoagulation activity versus normal human pool plasma as observed in thechromogenic activity assay (Table 33). The activity percentage ofFVII-CTP₄ is much higher compared to activity percentage of FVII-CTP₃and FVII-CTP₅. Methodological limitations of the ELISA method may limitthe accuracy of Ag level calculations of FVII-CTP₄.

5.8 Pharmacokinetic Study

Two pharmacokinetic studies were performed in order to determine theFVII-CTP₃, FVII-CTP₄, and FVII-CTP₅ pharmacokinetics (PK) parameters.During the first study, FVII-CTP₃, FVII-CTP₄, and FVII-CTP₅ (Group A, Band C, respectively) were administered in a single intravenous injectionto Sprague Dawley rats (six rats per treatment) in a dose of 250 μg/kgbody weight. Blood samples were drawn retro-orbitally from 3 ratsalternately at 0.083, 0.5 2, 5, 8, 24, 48, 72 and 96 hours post-dosing(Table 34). Citrated plasma (0.38%) was prepared immediately aftersampling and stored at −20° C. until analysis.

TABLE 34 Pharmacokinetic Study Design - Concentrated Harvest No. of DoseLevel Injected Treatment Test animals/group/ Dose (μg per Vol. Conc.Time-Points Group Article time point Route animal) (μl) (μg/ml) (hourspost-dose) A FVII- 6 IV 50 200 250 0 (Pre-dose) 0.083, 0.5, CTP*3 2, 5,8, 24, 48, 72, 96 B FVII- 6 IV 50 200 250 0 (Pre-dose) 0.083, 0.5, CTP*42, 5, 8, 24, 48, 72, 96 C FVII- 6 IV 50 200 250 0 (Pre-dose) 0.083, 0.5,CTP*5 2, 5, 8, 24, 48, 72, 96

FVII concentration in plasma samples were quantified using human FVIIElisa kits (Zymutest FVII-Biophen). The pharmacokinetic profile wascalculated and is the mean of 3 animals at each time point. Terminalhalf-life values were calculated using PK Solutions 2.0 Software. Table35 below summarizes the calculated FVII concentrations at the differentsampling time points. The PK profile (FIGS. 23-24) and a summary of thePK parameters (Table 36) are also presented below. FVII-CTP₅demonstrated a superior profile as compared to FVII-CTP₃ and FVII-CTP₄(Table 36).

TABLE 35 First Pharmacokinetic Study - FVII Concentrations AVE-FVII-AVE-FVII- AVE-FVII- Time 3-CTP 4-CTP 5-CTP (hr) (ng/ml) SD (ng/ml) SD(ng/ml) SD 0.083 4214 583 3600 427 4888 504 0.5 3386 892 5213 1682 53842549 2 1138 219 3603 1338 3082 289 5 1390 374 2726 1127 2480 561 8 333167 1349 44 2316 633 24 133 12 476 98 788 34 48 38 3 165 24 384 61 72 122 91 62 167 31 96 26 1 42 8 93 49

TABLE 36 Pharmacokinetic Analysis FVII- FVII 3-CTP FVII-4-CTP 5CTPhalf-life (0.083-8 hr) (hr) 2.5 4.9 6.6 half-life (8-72 hr) (hr) 13.316.6 17.7 AUC (ng-hr/ml) (8-72 hr) 18374.6 51224.4 72954.2 Vd (ml/kg)(8-72 hr) 203.7 91.9 67.7 CL(ml/hr/kg) (8-72 hr) 10.6 3.8 2.7

The addition of four or five CTPs significantly elongated FVII half-lifeas compared to 3 CTPs by 2- and 3-fold, respectively (Table 36). Thissuperiority was more significant in the initial part of the study(0.083-8 hr), suggesting a potential improved protein recovery andreduced extra vascular clearance. AUC following FVII-CTP₄ and FVII-CTP₅administration increased by 3- and 4-fold, respectively, versusFVII-CTP₃. Clearance was also reduced while adding 4 and 5 CTPs to FVII(Table 36).

As observed in the study, the addition of four and five CTPssignificantly elongated FVII half-life as compared to 3 CTPs, both inthe initial and terminal half-life. The half-life values in the firstand second study are different due to a different analysis approachwhich was effected by the dose and study duration, nevertheless theoverall trend was maintained. The AUC following FVII-CTP₄ and FVII-CTP₅administration increased by 2.5- and 7-fold, respectively, versusFVII-CTP₃.

5.9 Conclusions:

In this study, the PK parameters and potential clotting activity ofFVII-CTP₃, FVII-CTP₄, and FVII-CTP₅ were assessed. Fusion of 4 and 5CTPs to FVII provided a superior and improved half-life, exposure andreduced clearance as compared to FVII-CTP₃ while maintaining a similarchromogenic and in vitro clotting activity. These results were observedat different concentrations of protein and were consistent for bothharvest and purified protein. While evaluating the overall effect offusion of CTP at the C terminus to FVII, fusion of 1-5 CTPs considerablyincreased the half-life and AUC of FVII in a CTP proportional manner,suggesting that as the CTP portion of the molecule increases, FVIIlongevity and stability is significantly improved while maintaining itsinitial in vitro clotting activity, as summarized in Table 37hereinbelow.

TABLE 37 Comparative assessment T_(1/2) percent increase AUC percentincrease FVII vs. FVII-CTP₂ 268 200 FVII-CTP₂ vs. FVII-CTP₃ 67 57.8FVII-CTP₃ vs. FVII-CTP₄ 24 178 FVII-CTP₄ vs. FVII-CTP₅ 6 42

As previously reported, FVII half-life correlates with the half-life ofthe activated form of FVII (FVIIa) both in humans and animals.Therefore, it is anticipated that a similar improvement in half-lifewill be obtained for the activated versions following CTP fusion.

Example 6 FVII-CTP₃ Feasibility Studies in FVIII-deficient HemophilicMice

Studies described hereinabove testing FVII-CTP, FVII-CTP₂ and FVII-CTP₃harvest PK profile and coagulation activity vs. a commercial FVII wereconducted. FVII-CTP₃ exhibited an improved PK profile while maintainingits coagulation activity vs. FVII-CTP and FVII-CTP₂ harvests or rhFVII.In order to further characterize FVII-CTP₃ in vitro and in vivoproperties, a mini stable pool expressing and secreting the protein wasgenerated, and purification and activation processes were developed.

In the current study, the pharmacokinetic and pharmacodynamic propertiesof FVIIa-CTP₃ were tested in FVIII-deficient mice. The PK profile of theprotein was evaluated. A FVIIa specific activity-based PK profile wasestablished and compared to commercial product NovoSeven®. In addition,the long-lasting in vivo hemostatic capabilities of FVIIa-CTP₃ to inducecoagulation in FVIII-deficient mice after a tail vain transection(survival study) were tested.

Study Objectives:

To evaluate the pharmacokinetic and pharmacodynamic parameters ofFVIIa-CTP₃ vs. commercial rhFVIIa (NovoSeven®) in FVIII-deficient micefollowing a single IV administration at a similar activity dose.

To determine the in vivo ability of FVIIa-CTP₃ to maintain homoeostasisin FVIII-deficient mice by a single IV administration of FVIIa-CTP₃ andNovoSeven® at a similar activity dose followed by a challenge of tailvein transection (survival study).

Production of FVII-CTP₃ Harvest:

FVII-CTP₃ was expressed in-house in Dg44 cells using a pCI-DHFR vector.Stable transfected pool #71 was grown in shake flasks, in the presenceof 25 ng/L of Vitamin K3 (Sigma). Cell suspension was cultured andharvested following viability decline to 60-80%. The harvest wasfiltered and frozen at −70° C.

Determination of Harvest FVII Antigen Level:

FVII antigen level was determined using human FVII ELISA kit (ZymotestHyPhen) (Table 38). The antigen level was calculated per each pooledharvest batch.

TABLE 38 FVII-CTP₃ antigen level FVII antigen level PK-PD study Survivalstudy harvest 31A harvest 31B harvest 38 Av (μg/ml) 16.0 15.9 16.6 STD1.5 0.0 0.8 % CV 9.1 0.1 4.9

FVII-CTP₃ Purification Process (FIG. 25)

Process Outline

Following a short purification study, the following purification processusing 2 columns was performed. VII-Select affinity column (GE) andCeramic Hydroxyapatite type 1 (HA), 40 μm (Bio Rad), FVII-CTP₃γ-carboxylated enriched protein was purified. Auto-activation wasinduced by incubation of purified FVII-CTP₃ in the presence of CaCl₂overnight at 2-8° C. The purification process is in its finaldevelopmental stage and is being optimized, thus part of thepurification steps are not identical in the two batches.

Ultra-filtration/diafiltration (UFDF) using 10 kDa Hollow Fiber orPellicon Cassette

Clarified harvest was thawed at 4° C. over the weekend (2-3 days).

In Batch 31, clarified harvest (12 liters) was concentrated 4-fold (intwo successive runs) using a hollow fiber cartridge (GE HealthcareCatalog #UFP-10-C-4X2MA) with a 10 KDa molecular weight cut-off.Concentrated harvest was dia-filtrated against 1-2 volumes of TBS (50 mMTris 150 mM NaCl pH 7.4).

In Batch 38, clarified harvest (8.5 liters) was concentrated 4-foldusing a Pellicon 2 (Millipore) cassette with a 10 KDa molecular weightcut-off. Concentrated harvest was directly loaded on VII-Select column.

Both ultra-filtrations were performed on ice with ice cold buffers. UFDFsamples were filtered 0.22 μm before loading.

Capture on FVII-Select Column

The UFDF or concentrated harvest was loaded on VII-Select column(XK16/20, CV 18 ml), pre-equilibrated with TBS pH 7.4. The column waswashed with 50 mM Tris-HCl, 0.5M NaCl pH 7.5, and FVII-CTP₃ was elutedwith 50 mM Tris-HCl, 1M-NaCl 50% (v/v), Propylene Glycol pH 7.5. Theprocess was performed in two successive cycles utilizing the samecolumn.

Gamma Carboxylation-Based Separation on a Ceramic Hydroxyapatite Column

The eluted product was diluted 1:10 with 10 mM sodium phosphate pH 6.8and loaded on ceramic hydroxyapatite columns (XK16/20, CV 24 ml). Thecolumn was washed with 59 mM sodium phosphate pH 6.8 and theγ-carboxylated rich fraction of Factor VII was eluted with 500 mM sodiumphosphate pH 6.8. This process was performed in two successive cycles onthe same column. At each batch, the eluates of the two cycles werepooled and concentrated to 1.7-2 mg/ml and dia-filtered with 20 mMTris-HCl, 100 mM NaCl pH 8.2 to reduce volume and prepare the materialfor the activation step.

FVII Activation

Purified FVII-CTP₃ was diluted to 1 mg/ml and incubated in 20 mMTris-HCl, 100 mM NaCl and 1 mM CaCl₂ pH 8.2 at 2-8° C. for 24 hours.Activation was terminated by buffer exchange (UFDF) to preliminaryformulation buffer (20 mM Citrate, 240 mM NaCl, 13.3 mM Glycine, pH6.9).

FVII-CTP₃ and FVIIa-CTP₃ Analytical Properties:

SDS-PAGE and Western Blots

Purified FVII-CTP₃, and FVIIa-CTP₃ were loaded on 12% Tris-Glycine gelusing Precision Plus Dual Color Protein Marker (Bio-Rad). The SDS-PAGECoomassie analysis was performed by staining the gel with Coomassiebrilliant blue reagent (5 or 10 μg of protein/lane). Western blotanalysis was performed (1 μg of protein/lane) using anti-human FVIIpolyclonal Ab (R&D systems; AF2338), anti-human gamma carboxylationmonoclonal antibody (American Diagnostics Catalog #499, 3570), andanti-CTP polyclonal Ab. Under reduced conditions, FVII-CTP₃ migrated at75 KDa, and FVIIa-CTP₃ migrated as two main bands: a heavy chain at 50kDa, and a light chain at 25 kDa, represented in FIG. 26 as Bands 2 and3, respectively.

The purification procedure significantly enriched the FVII-CTP₃ portionwhile reducing impurities. The purification process yield was 25-30%FVII (according to ELISA). Most of the protein lost during purificationhad low FVII chromogenic activity or no activity. Based onCoomassie-stained SDS-PAGE, the reduced FVIIa-CTP₃ contains more thanthe predicted bands. A band migrating to around ˜75 kDa representsnon-activated FVII (FIG. 26, Band 1). This band consists of two bandswith minor MW differences, which might reflect different γ-carboxylationcontent. Additional bands with MW lower than 20 kDa were observed. Thiswas previously reported to be degradation products of the heavy chain.

FVII-CTP₃ Chromogenic Activity:

A comparative assessment of the in vitro potency of FVII-CTP₃ harvest,in-process fractions, and purified FVII-CTP₃ versus human pool normalplasma was performed using a commercially available chromogenic activitytest kit, BIOPHEN (Hyphen BioMed 221304). FVII-CTP₃ harvest and proteinwere serially diluted and the potency was assessed by comparing adose-response curve to a reference preparation of normal human plasma.Following FVII-CTP₃ purification, the chromogenic activity wassignificantly improved, and non-active fractions were separated mainlyby HA column (FIG. 27). A strong correlation between FVII chromogenicactivity and detection of FVII with monoclonal anti-Gla antibodies inWestern blot was observed. The potency of FVII chromogenic activity asreflected by EC50 value in harvest is affected from both carboxylatedand non-carboxylated FVII fractions. Following purification andenrichment of FVII-CTP₃ γ-carboxylated fraction, the activity wasimproved, demonstrating the important contribution of γ-carboxylation toFVII activity (FIG. 27). This parameter is crucial for proper FVII invivo activity and will be further addressed in a clone developmentprogram.

Protein Determination by A280

The theoretical extinction coefficient of FVIIa-CTP₃ and NovoSeven® wascalculated using the ProtParam algorithm(http://web.expasy.org/protparam). The calculation is based on aminoacid sequence. The calculated extinction coefficients for FVII-CTP₃ andNovoSeven® is 1.186 and 1.406, respectively. These values represent theabsorbance of 1 g/L at 280 nm.

The extinction coefficient difference between the two proteins derivessolely from the increase in molecular weight of FVIIa-CTP₃ compared toNovoSeven®, since CTP lacks aromatic and cysteine residues, thus doesnot contribute to the absorbance.

Protein determination by A280 is used for final FVII, and for purifiedin-process samples, starting from the elution of VII-Select column.

Determination of FVIIa Antigen Level

FVIIa antigen level was determined using Human FVIIa ELISA kit (IMUBIND,American Diagnostica). The antigen level was calculated per each batch.However, this tool was not useful for the determination of the dose forinjection, since it did not represent the amount of active product.

Clotting assay of FVIIa-Staclot® VIIa-rTF

FVIIa is derived from an intra-chain cleavage of the single-chain FVII.Native tissue factor (TF) is a cofactor of FVIIa. Upon binding to TF,FVII mediates the activation of Factor X to Xa, while itself istransformed to FVIIa. The soluble tissue factor is the extracellularpart of native tissue factor. It can no longer activate FVII byauto-activation, but the FVIIa bound to tissue factor can activate FX toFXa.

The recombinant soluble tissue factor (rsTF) used in this assay utilizesthe FVIIa specificity to construct a FVIIa clotting test. rsTF, in thepresence of FVIIa, calcium and phospholipids leads to coagulation ofplasma, without activating FVII to FVIIa.

The observed clotting time in this system has an inverse relationshipwith the FVIIa content in the tested sample, with no interference ofFVII presence in the sample.

The assay was performed by Omri Laboratories (Nes-Ziona, Israel). FVIIaactivity was evaluated for both NovoSeven® following reconstitution andFVIIa-CTP₃ prior to each study. NovoSeven® activity did not correlatewith the anticipated activity as reported on the vial, but thediscrepancy might be due to a different approach for activityevaluation. Table 39 summarizes the FVIIa clotting activity per volumewithout considering the protein concentration.

TABLE 39 FVIIa clotting activity of batch products PK study SurvivalStudy FVIIa- FVIIa- 3 * CTP 3 * CTP (FVIIa 31) NovoSeven ® (FVIIa 38)NovoSeven ® Activity (U/ml) 1.3E+06 2.5E+05 1.3E+06 7.4E+05

Specific Activity of FVIIa-CTP₃

FVIIa specific activity (which is calculated as the activity/ml dividedby protein concentration) was calculated based on A280 and is presentedin Table 40. When comparing the specific activity of the two molecules,which differ in MW, compensation must be made in order to normalize theactivity (i.e. because of the molecular weight difference, the number ofactive sites in 1 mg of NovoSeven® is 1.185-fold higher than inFVIIa-CTP₃). Calculation of the conversion factor is presented in thefollowing equation:

$\begin{matrix}{{Normalized\_ SA} = {{\frac{{SA}\left( {{FVIa}\text{-}{CTP}_{3}} \right)}{{MW}.\left( {{FVII}\mspace{14mu} {CTP}_{3}} \right)} \times {{MW}({Native\_ FVII})}} =}} \\{= {\frac{{SA}\left( {{FVIIa}\mspace{14mu} {CTP}_{3}} \right)}{53419.5\; {Da}} \times 45079.1\; {Da}}} \\{= {{{SA}\left( {{FVIIa}\text{-}{CTP}_{3}} \right)}^{*}1.185}}\end{matrix}$

TABLE 40 FVIIa-CTP₃ specific activity compared to NovoSeven ® SpecificFold Prot Activity decrease Average STDV Extinction conc. U/mg U/mg fromSample A280 (n = 9) % CV coefficient (mg/ml) U/ml protein FVIIaNovoSeven ® NovoSeven ® 1.274 0.031 2.398 1.406 0.906 8.36E+05 9.23E+059.23E+05 1.0 FVIIa-CTP₃ 4.396 0.092 2.094 1.186 3.706 7.23E+05 1.95E+052.31E+05 4.0

FVIIa-CTP₃ PK-PD Study:

Study Outline

FVIIa-CTP₃ and rhFVIIa (NovoSeven®, NS) were administered in a singleintravenous injection to C57B FVIII-deficient mice at a dose of 6.4E6U/kg body weight (160,000 U/animal). Blood samples were drawnretro-orbitally from 4 mice alternately at 0.166, 0.5, 2, 4, 8, 12, 24,34, 48, 58, and 72 hours post-dosing (Table 41). Citrated plasma (0.32%)was prepared immediately after sampling and stored at −20° C. untilanalysis. FVIIa clotting activity level was evaluated, and a detailed PKanalysis was performed. The study was performed by Omri Laboratories(Nes-Ziona, Israel).

TABLE 41 Study outline No. of animals Amount Injected Treated Testgroup/ Dose of Units/ Vol. Time-Points Groups Article timepoint Routeanimal (μl) (hours post-dose) A rhFVIIa 4 IV 1.6e5 200 0 (Pre-dose)0.166, 0.5, 2, 4, 8, 12, 24, 34, 48, 58, 72 B FVIIa- 4 IV 1.6e5 200 0(Pre-dose) 0.166, 0.5, 2, CTP₃ 4, 8, 12, 24, 34, 48, 58, 72

FVIIa-CTP₃ PK Profile in FVIII-deficient Mice

FVIIa activity in blood samples was quantitated using a Staclot®VIIa-rTF kit (Stago, Parsippany, N.J.). The pharmacokinetic profile wascalculated for each protein and represents the mean of 4 animals at eachtime point. FIG. 28 presents the PK profile of FVIIa throughout theexperiment. FVIIa recovery is presented in Table 43. A summary of the PKparameters is presented in Table 44.

Table 42 summarizes the clotting activity values followingadministration of either NovoSeven® or FVIIa-CTP₃. FVIIa-CTP₃ andNovoSeven® reached maximal activity half an hour post-dosing.NovoSeven®'s highest activity value reached only 43% of FVIIa-CTP₃'smaximal activity value. FVIIa-CTP₃ clotting activity was maintained fora longer period of time, demonstrating elongated activity. Clottingactivity for the NovoSeven®-treated mice was undetectable at time pointslater than 12 hours, while FVII-CTP₃ treated mice continued to retainmeasurable activity at 48 hours post dosing (Table 42 and FIG. 28).

The addition of three tandem CTP copies to FVIIa elevated recovery by100% (Table 43), as measured by the highest activity post-dosing andcompared to the anticipated activity based on in vitro analysis, andincreased the half-life and mean resident time (MRT) 5-fold. Theexposure time (AUC) was increased 3-fold (Table 44).

TABLE 42 FVIIa clotting activity following single IV injection Timeafter administration Average FVIIa Clotting Activity (U/ml) (hours)FVIIa-CTP₃ NovoSeven ® 0.16 6.8E+07 3.2E+07 0.5 9.7E+07 4.3E+07 22.1E+07 3.9E+06 4 7.7E+06 7.3E+05 8 2.7E+06 4.2E+04 12 3.7E+05 6.2E+0324 2.4E+04 BLQ 34 4.6E+03 BLQ 48 1.5E+03 BLQ

TABLE 43 FVIIa-CTP₃ recovery Practical *Anticipated Treated. Test Amountof administered Cmax Cmax % Groups Article Units/animal dose (U/ml)(U/ml blood) (U/ml) Recovery A rFVIIa 1.60E+05 1.20E+06 1.40E+054.25E+04 30 B FVIIa- 1.60E+05 1.29E+06 1.50E+05 9.74E+04 64.6 CTP₃*anticipated Cmax is derived from administered dose divided in bloodvolume

TABLE 44 PK parameters of FVIIa-CTP₃ vs. NovoSeven ® PK ParametersNovoSeven ® FVIIa-CTP₃ Half-life-_(α) (0.5-12 hr) 0.94 1.57Half-life-_(β) (12-48 hr) NA 4.62 AUC (mU * hr/ml) 5.80E+07 1.80E+08Vd/Kg (ml/Kg) 1408 2375 CL/Kg (ml/hr/Kg) 1034 356 MRT (hr) 1.3 6.7

Thrombin Generation Assay (TGA)

The generation of thrombin is a fundamental part of the clotting cascadeand as such an estimate of how well a particular individual can generatethrombin may correlate with either a risk of bleeding or thrombosis.Commonly measured variables when analyzing thrombin generation include:the lag time, the time to peak thrombin generation, the peak, theendogenous thrombin potential [ETP] (i.e., the area under the curve andthe tail), the time course of the thrombogram (“TG”). After a lag time,a burst of thrombin is observed. However, clotting occurs at the end ofthe lag time, when more than 95% of all thrombin has not yet formed. Thethrombin generation assay was performed at Omri Laboratories, usingThrombinoscope reagents supplemented with human hemophilic plasma. TGAreflects of the clotting ability in mice plasma, derived from injectionof NovoSeven® and FVIIa-CTP₃. FIG. 29 presents TGA parameter values formice plasma following administration of either FVIIa-CTP₃ or NovoSeven®.Following FVIIa-CTP₃ administration, all three parameters (rate ofthrombin generation, maximal amount of generated thrombin and KIIa)demonstrate an advantage of FVII-CTP₃ over NovoSeven® treatment. Thisfurther strengthens the notion of potential long-acting superiority ofFVII-CTP₃ as compared to NovoSeven®.

FVIIa-CTP₃ Tail Vain Transection (TVT) Study:

Study Outline

The data obtained from the PK/PD test for FVIIa-CTP₃ provided insightinto the functionality of FVIIa-CTP₃, and demonstrated that FVIIa-CTP₃had a pharmacokinetic advantage when compared with NovoSeven®. However,the ability of the protein to induce a clot in vivo, after a traumaticevent has not yet been demonstrated. In order to evaluate the ability ofFVIIa-CTP₃ to stop bleeding, the same FVIII-deficient mice model wasemployed for a bleeding challenge.

FVIII-deficient mice were administered a single intravenous injection ofFVIIa-CTP₃ or NovoSeven®. The mice were dosed with drug in amounts thatprovided equivalent FVIIa activity (1.6E05 units, 200 μl), calculatedaccording to the potency of each drug evaluated in the FVIIa clotactivity assay (Table 45). The administered doses were 9 mg/kg ofNovoSeven®, and 40 mg/kg of FVII-CTP₃ due to the reduced activity ofFVIIa-CTP₃. A control group was injected with 200 μl vehicle.

The tail vein was transected 2.7 cm from the tail tip 15 min (injection1), 24 hours (injection 2) or 48 hours (injection 3)post-administration, and mice survival was recorded for 24 hours.

TABLE 45 Evaluation of injected samples NovoSeven ® FVIIa-CTP₃ proteinSpecific protein Specific Specific Injection conc. Activity Activityconc. Activity Activity Activity No. (mg/ml) (U/ml) (U/mg) (mg/ml)(U/ml) (U/mg) (normalized) 1 0.91 8.0E+05 8.8E+05 3.63 6.6E+05 1.8E+052.2E+05 2 0.92 8.3E+05 9.0E+05 3.81 7.8E+05 2.0E+05 2.4E+05 3 0.898.8E+05 9.9E+05 3.68 7.3E+05 2.0E+05 2.3E+05

Protein concentration was determined by A280.

Results

Data from the vehicle-injected control groups for the three injections(5 animals×3 injections), were summarized and are presented in FIG. 30.30% survival was observed 24 hours after tail vein transection.

NovoSeven® and FVIIa-CTP₃-treated mice demonstrated proper hemostaticactivity after tail vein transection performed 15 min after FVIIaadministration. A 100% survival rate was observed in FVIIa-CTP₃ andNovoSeven® treated animals (FIG. 30).

The reduced clearance rate of FVII-CTP₃ which was demonstrated in thePK/PD study is most clearly appreciated after a tail vein transectionperformed 24 hours post-administration. A decline in the survival rateof NovoSeven® is observed. Similar to the control group, 50% death isobserved within 10 hours. Meanwhile, 90% of FVIIa-CTP₃ treated micesurvived (FIG. 30). This result emphasizes the long-lasting efficacy ofthe FVIIa-CTP₃ treatment.

48 hours after administration, a decline in survival rate isdemonstrated in groups treated with either FVIIa-CTP₃ or NovoSeven®(FIG. 30C). A slight improvement in FVIIa-CTP mice was observed, but thedifference did not reach statistical significance.

Discussion:

CTP fusion to recombinant proteins extends the circulatory half-life ofproteins while maintaining comparable activity. While the mechanismbehind the reduced clearance of protein above a threshold size of 70 KDais well understood with respect to renal clearance, additionalprotection is achieved following CTP fusion. CTP fusion is believed tosweep around the protein shield and protect it from proteolyticcleavage, to increase its radial molecular weight due to the highlynegative charge and to reduce its affinity to hepatic clearancereceptors.

The present study was aimed to provide specific insight on the impact ofCTP fusion to FVII on protein half-life and clearance and also addressthe paradigm of its specific activity following this modification.FVIII-deficient mice were administered with a single IV injection ofFVIIa-CTP₃ or recombinant commercial FVIIa (NovoSeven®) at similar dose(unit based) and a PK activity-based analysis was performed. FVIIa-CTP₃demonstrated a superior longevity as reflected by 5- and 3.5-foldincrease in its half-life and AUC, respectively. The specific activity(U/mg) of FVIIa-CTP as calculated by the Staclot® activity kit dividedby the protein concentration measured by A280 was shown to be 4-5 timeslower than the specific activity of NovoSeven®.

To build on the understanding of how CTP affects the haemostatic effectsof FVIIa in vivo, the ability of FVIIa-CTP₃ to reduce bleeding wasinvestigated. In the tail vein transection bleeding model in hemophilicmice model, rFVIIa administration can improve the survival rate ofchallenged animals and avoid their bleeding to death. In the studydescribed herein, animals were administered with FVIIa-CTP₃ orNovoSeven®. Both molecules were able to maintain homeostasis when thetransection was performed 0.25 hours post-dosing. A significantlyprolonged duration of activity was demonstrated for theFVIIa-CTP₃-treated group when the tail transection was performed 24 hrpost dosing. The vehicle-treated group's survival rate was higher thananticipated and higher than that obtained in previous studies (50% vs.20% in previous studies, data not shown). The percent survival oftreated animals at is further evaluated at earlier time points,including at 36 hr post dosing.

In conclusion, it was demonstrated that FVIIa-CTP₃ has an increasedduration of activity in hemophilic mice which translates into a longerduration of haemostatic effect when compared to NovoSeven®. The datagathered suggest that fusion of CTP to FVII is a technology with thepotential to significantly improve prophylactic treatment in patientswith hemophilia.

Example 7 Comparative Assessment of Purified FVII-CTP₃ vs. FVII-CTP₅Profile Following Single IV or SC Injection to SD Rats Study Objective

Two studies were carried out:

The first study objective was to determine the pharmacokineticparameters of rFVII-CTP3 versus rFVII-CTP5 following FVII select- andHA-column purification in male Spargue Dawley rats, after a singleintravenous administration of 50 μg/animal.

In the second study, rFVII-CTP3-HA versus rFVII-CTP5-HA pharmacokineticparameters, were examined in male Spargue Dawley rats following a singleintravenous or subcutaneous administration of 100 μg/animal.

Results

Determination of FVII-CTP 3 and FVII-CTP 5 Antigen Level

FVII antigen level was determined using Human FVII ELISA kit (ZymotestHyPhen) (Table 46). T

TABLE 46 Summarizes the calculated protein concentration which is theaverage of three independent runs. FVII 5 CTP FVII 3 CTP FVII HA 5 FVIIS46 el. FVII HA 46 el. FVIIS el. 100% B Conc. Dial Conc. Dial Conc. DialConc. Dial AVE (ng\ml) 3.78E+06 1.59E+06 1.88E+06 7.92E+05 SD 1.30E+066.03E+05 7.15E+05 3.57E+05 CV (%) 3.43E+01 3.80E+01 3.80E+01 4.51E+01

Western Blot Analysis of the Examined Samples

FVII-CTP_(3, 5) samples were loaded on 4-12% bisTrisgel (NuPage,invitrogene) using Precision plus dual color protein marker (Bio-Rad).The SDS-PAGE analysis was performed by western immune-blot usingpolyclonal anti FVII Ab (R&D systems), anti CTP polyclonal Ab (Adarbiotech production) or anti Gla Ab (American diagnostica). In summary,FVII fused to three and five CTP migrated at 80 and 100 kDa,respectively (see FIG. 31).

Comparative Assessment of FVII In Vitro Potency

FVII activity assay, which was performed in Sheba medical center, thenational coagulation center, is a PT based assay using immunoadsorbedplasma deficient in factor VII (Siemens). The PT reagent is innovin andthe assay is performed in the Sysmex CA 1500 instrument. FVII normalrange is within 55-145%. Sample activities are summarized in Table 47.

TABLE 47 Sample activity Concentration Concentration (mg/ml) in testedAverage- according to sample Results % of Sample (NANODROP) (μg/ml) (%)plasma FVII-5CTP 2.19 2 87 16% FVIIS el. 1 30 Conc. Dial 0.5 10FVII-5CTP HA 5 1 2 97 21% 100% B 1 36 conc. Dial 0.5 13 FVIIS 46 el.3.17 2 100 18% Conc. Dial 1 35 0.5 12 FVII HA 46 el. 1.5 2 92 20% Conc.Dial (1) 1 33 0.5 10

The normal level of circulating FVII in the body is around 0.5 μg/ml.Both, FVII-CTP₃ and FVII-CTP₅ exhibit about 5 fold reductions in theircoagulation activity versus normal human pool plasma.

Pharmacokinetic Study

Two pharmacokinetic studies were performed in order to determine theFVII-CTP₃ and FVII-CTP₅ (after FVII select and FVII HA column)pharmacokinetics (PK) profile and parameters. In the first study,FVII-CTP₃, and FVII-CTP₅ following FVII select/HA purification wereadministered in a single intravenous injection to Spargue Dawley rats(six rats per substance) in a dose of 50 μg/rat.

Blood samples were drawn retro-orbital from 3 rats alternately at 0.083,0.5 2, 5, 8, 24, 48, 72, 96 and 120 hours post dosing. Citrated plasma(0.38%) was prepared immediately after sampling and stored at −20 untilanalysis.

In the second study, only samples after HA column were tested. Thesesamples were administered in a single intravenous or subcutaneousinjection to Spargue Dawley rats (six rats per substance) using a doseof 100 μg/rat. Blood samples were collected at the same time points andconditions as at the first study above.

TABLE 48 First study design (FVII select vs. FVII HA). No. of Dose LevelInjected Treated animals/ Dose (μg per Vol. Conc. Time-Points GroupsTest Article group/ Route animal) (μl) (μg/ml) (hours post-dose) AFVII-CTP*3 6 IV 50 200 250 0 (Pre-dose) batch 46 HA 0.083 0.5, 2, 5, 8,24, 48, 72, 96, 120 B FVII-CTP*3 6 IV 50 200 250 0 (Pre-dose) batch 460.083 0.5, 2, 5, 8, FVIIS 24, 48, 72, 96, 120 C FVII- 6 IV 50 200 250 0(Pre-dose) CTP*5batch 0.083 0.5, 2, 5, 8, 5 HA 24, 48, 72, 96, 120 DFVII-CTP*5 6 IV 50 200 250 0 (Pre-dose) batch 5 0.083 0.5, 2, 5, 8,FVIIS 24, 48, 72, 96, 120

TABLE 49 Second study design (IV vs. SC) No. of Dose Level InjectedTreated animals/ Dose (μg per Vol. Conc. Time-Points Groups Test Articlegroup/ Route animal) (μl) (μg/ml) (hours post-dose) A FVII- 6 IV 100 200500 0 (Pre-dose) CTP*3 0.083 0.5, 2, 5, 8, batch 46 HA 24, 48, 72, 96,120 B FVII- 6 SC 100 200 500 0 (Pre-dose) CTP*3 0.083 0.5, 2, 5, 8,batch 46 HA 24, 48, 72, 96, 120 C FVII- 6 IV 100 200 500 0 (Pre-dose)CTP*5batch 0.083 0.5, 2, 5, 8, 5 HA 24, 48, 72, 96, 120 D FVII- 6 SC 100200 500 0 (Pre-dose) CTP*5 0.083 0.5, 2, 5, 8, batch 5 HA 24, 48, 72,96, 120

The main differences between these two studies are the dosages and theroute of administration. In the first study, rats were injected IV with50 μg\rat, while in the second study, the rats were injected IV or SCwith 100 μg\rat (total 500 μg/kg; rats weigh 200 g). The increase in thedosage is due to the change in the type of administration; SCadministration requires higher amounts to achieve effects similar to IVadministration.

Analysis of PK Study

FVII concentration in plasma samples were quantified using human FVIIElisa kits (zymutest FVII-Biophen). Pharmacokinetic profiles werecalculated and reflect the mean for 3 animals at each time point.Terminal half-live values were calculated using PK solutions 2.0software. The table below summarizes the calculated FVII concentrationsat the different sampling time points. PK profile and a summary of thePK parameters are presented in table below.

TABLE 50 First pharmacokinetic study (FVII select vs. FVII HA) -FVIIconcentrations (ng\ml). FVII CTP * 3 FVII CTP * 3 FVII CTP * 5 FVIICTP * 5 Time BATCH 46 BATCH 46 BATCH 5 BATCH 5 (hour) HA FVII S HA FVIIS 0.083 1816.3 1633.9 2064.3 1853.5 0.5 1523.7 1409.9 1351.4 1418.0 21284.9 1041.7 1389.7 834.4 5 607.9 531.6 722.7 737.2 8 524.2 430.0 712.2614.6 24 115.5 132.9 272.5 201.8 48 21.1 31.6 62.3 90.4 72 9.5 15.8 29.131.8 96 BLQ 5.8 7.0 16.9 120 BLQ BLQ 8.5 13.4

TABLE 51 Second pharmacokinetic study (IV vs. SC) -FVII concentrations(ng\ml). FVII FVII CTP * 3 FVII CTP * 5 CTP * 3 FVII CTP * 5 Time BATCH46 BATCH 5 BATCH 46 BATCH 5 (hour) HA-IV HA-IV HA-SC HA-SC 0.083 6452.66153.3 5.0 BLQ 0.5 3930.7 3660.6 14.5 14.6 2 1992.3 2176.2 113.6 96.2 51598.9 2087.3 106.6 70.5 8 781.6 1075.6 188.9 129.7 24 268.5 627.2 155.0239.2 48 51.9 143.3 43.0 88.6 72 8.8 39.0 7.0 36.7 96 BLQ 10.8 BLQ 10.4120 BLQ 8.2 BLQ 8.7

TABLE 52 PK Analysis- first pharmacokinetic study (FVII S vs. HA). FVIIFVII FVII FVII CTP * 3 CTP * 3 CTP * 5 CTP * 5 BATCH 46 BATCH 46 BATCH 5BATCH 5 HA FVII S HA FVII S half-life (0.083-8 hr) 4.3 4.0 5.51 5.59(hr) half-life 11.1 12.1 16.46 20.29 (8-72\96\120 hr) (hr) half-life(8-72) (hr) 11.1 13.4 13.62 15.64 AUC (O-t) 14566.9 13686.4 21812.719307.9 (obs area) (8-72/96/120 hr) AUC (∞) area 14718.2 13788.1 22013.919701 (8-72/96/120 hr) Vd (area)/kg (ml/kg) 271.1 316.1 269.7 371.5(8-2/96/120 hr) CL (area)/kg 17.0 18.1 11.356 12.69 (ml/hr/kg)(8-72/96/120 hr)

The addition of five CTP elongated FVII half-life compared to 3 CTPs.Both forms of 5 CTP (i.e FVIIS and FVII HA) were detected at the longtime points (96 and 120 hr), while FVII-3 CTP HA and FVIIS-3 CTP weredetected until 72 hr and 96 hr, respectively. Based on this fact, thehalf-life of FVII-5 CTPs is longer than 3CTPs variants (see FIG. 32).Comparing half-life of all examined materials (3 and 5 CTPs) at the sametime points (8-72 hr) showed that the half-life are similar, although 5CTP are quite longer (FIG. 32).

TABLE 53 PK analysis - second pharmacokinetic study-(IV vs. SC). FVIICTP*3 FVII CTP*5 FVII CTP*3 FVII CTP*5 BATCH BATCH BATCH BATCHBioviability Bioviability 46 HA-IV 5 HA-IV 46 HA-SC 5 HA-SC CTP*3 CTP*5half-life 3.0 3.9 −1.8 −3.18 (0.083-8 hr) (hr) half-life 9.9 14.6 13.1422.94 (8-72/96/120 hr) (hr) half-life 9.9 13.0 13.14 29.47 (8-72) (hr)AUC(O-t)(obs 28866.8 43761.0 6600 9822.7 22.9 22.4 area)(8-72/96/ 120hr) AUC (∞) 28993.0 43934.4 6733 10110.8 23.22 23.01 area(8-72/96/ 120hr) Vd(area)/kg 246.4 240.5 1407.6 1636.8 (ml/kg)(8-72/ 96/120 hr)CL(area)/kg(ml/ 17.2 11.4 74.261 49.452 hr/kg) (8-72/96/ 120 hr)

Again, as observed in the first study, the addition of 5 CTPs elongatedFVII half-life as compared to adding 3 CTP, both in the initial andterminal half-life and in both administration ways (IV and SC, see FIG.33). As expected, following SC administration, FVII was first detectedin the blood at a later time point as compared to when it wasadministered IV.

In the above, two PK studies were summarized. The main purpose of thefirst study was to check the difference between FVII-3CTP and FVII-5 CTPafter 2 different columns: FVII select and FVII HA. In our previousstudies, harvest vs. purified proteins were checked and it was foundthat the difference between 3 and 5 CTP versions of FVII was greaterwhen harvest was injected to the rats.

There was no significant difference between the results of FVII 3\5 CTPafter both columns, hence it was decided to inject FVII HA 3\5 CTP inthe second study (IV vs. SC).

Example 8 FVIIa-CTP₃ (MOD-5014) Survival Study in Fviii Deficient MiceFollowing Subcutaneous Injection Study Objective

To evaluate the efficacy of NovoSeven®, MOD-5014 (FVIIA-CTP₃) andMOD-5019 (FVIIA-CTP₅) in a tail vein transection study, followingsubcutaneous administration.

FVIIa-CTP₃(MOD-5014) and FVIIa-CTP₅(MOD 5019) Analytical Properties:

Protein Determination by A280

Theoretical extinction coefficient of NovoSeven® was calculated usingProtParam algorithm (http://web.expasy.org/protparam). The calculationis based on amino acid sequence. The calculated extinction coefficientfor NovoSeven® is 1.406, and for MOD-5019 is 1.075 (values represent theabsorbance of 1 g/L at 280 nm). Extinction coefficient of MOD-5014 wasdetermined by amino acid analysis at Mscan. The extinction coefficientsfor MOD-5014 is 1.27.

Clotting Assay of FVIIa-STACLOT VIIa-rTF

FVIIa is derived from intra-chain cleavage of the single-chain FVII.Native tissue factor (TF) is a cofactor of FVIIa, upon binding to TF,FVII mediates the activation of Factor X to Xa, while itself istransformed to FVIIa. The soluble tissue factor is the extra cellularpart of native tissue factor. It can no longer activate FVII by autoactivation, but the FVIIa bound to tissue factor can activate FX to FXa.

The recombinant soluble tissue factor (rsTF) used in this assay isutilizing the FVIIa specificity to construct a FVIIa clotting test.Recombinant soluble tissue factor (rsTF), in the presence of FVIIa,calcium and phospholipids, produces coagulation of plasma withoutactivating FVII to FVIIa.

The observed clotting time in this system has an inverse relationshipwith the FVIIa content in the tested sample, with no interference ofFVII presence in the sample.

FVIIa activity was evaluated for reconstituted NovoSeven®, and forMOD-5014 and MOD-5019 prior to each study.

FVIIa specific activity (which is calculated as the activity/ml dividedby protein concentration) was calculated based on A280 and is presentedin Table 54. When comparing specific activity of the two molecules,which differ in molecular weight, compensation must be made in order tonormalize the activity (i.e. because of the molecular weight difference,the number of active sites in 1 mg of NovoSeven® is 1.185 fold higherthan in MOD-5014 and 1.307 fold higher than MOD-5019). Hence,calculation of the conversion factor is presented in the followingformula:

$\begin{matrix}{{Normalized\_ SA} = {{\frac{{SA}\left( {{FVIa}\text{-}{CTP}_{3}} \right)}{{MW}.({Native\_ FVII})} \times {{MW}\left( {{FVII}\mspace{14mu} {CTP}_{3}} \right)}} =}} \\{= {\frac{{SA}\left( {{FVIIa}\mspace{14mu} {CTP}_{3}} \right)}{45079.1\; {Da}} \times 53419.5\; {Da}}} \\{= {{{SA}\left( {{FVIIa}\text{-}{CTP}_{3}} \right)}^{*}1.185}}\end{matrix}$

TABLE 54 MOD-5014 Specific activity compared to NovoSeven ® Protein Foldconc. By Specific Activity decrease from Sample A280 (mg/ml) (U/mgFVIIa) ®NovoSeven ®NovoSeven 0.93 52,487 1.0 MOD-5014 batch 73 1.425,490 2.05 MOD-5019 batch 9 3.0 11,698 4.48

Study Outline

The most significant measurement is the ability of the protein to inducea clot in vivo, after a traumatic event. In order to evaluate theability of MOD-5014 to stop bleeding, the same FVIII deficient micemodel was employed for a bleeding challenge.

FVIII deficient mice were administrated with a single subcutaneousinjection of MOD-5014, MOD-5019 or NovoSeven®. Group A and B were dosedwith NovoSeven® and MOD-5014 respectively, in equivalent amounts asFVIIa activity. Group C was dosed with MOD-5019 in equivalent amountFVIIa protein as MOD-5014, in order to evaluate the critical factor(activity or amount of protein). The administrated doses were 4.2 mg/kgof NovoSeven®, and 8.6 mg/kg of MOD-5014 and MOD-5019. The tail vein wastransected 2.7 cm from tail tip 12 hours post administration, and micesurvival was recorded for 24 hours.

TABLE 55 Group designation Administered Bleeding Dose time, mg InjectedNo. of hours Injection FVII/ Volume mice per post Group date TestArticle Kg mU/Kg (μl) group dosing A 13.1.13 ® NovoSeven 4.23 221,876100 10 12 B 15.1.13 MOD-5014, batch 73 8.59 218,750 160 10 12 C 27.1.13MOD-5019, batch 9 8.59 100,496 160 10 12

Results

The experiment data is summarized in Table 56- and in FIG. 34.

TABLE 56 TVT study results Time post No. of surviving mice % survivalTVT Novo- MOD- MOD- Novo- MOD- MOD- (h) Seven ® 5014 5019 Seven ® ® 50145019  0 9 10 10 100 100 100  1 9 10 10 100 100 100  2 9 10 10 100 100100  3 8 10 8 89 100 80  4 6 9 8 67 90 80  5 5 9 7 56 90 70  6 4 8 5 4480 50  7 3 8 5 33 80 50  8 2 7 5 22 70 50  9 1 6 5 11 60 50 10 1 5 5 1150 50 11 1 3 5 11 30 50 12 1 3 5 11 30 50 24 1 3 4 11 30 40

24 hours post TVT, only 11% of NovoSeven® injected mice have survived.30% of MOD-5014 and 40% of MOD-5019 have survived to this time point.Surprisingly, subcutaneously injected MOD-5014 and MOD-5019 showsimproved mice survival in comparison to NovoSeven®.

Factor VIIa, like other coagulation factors, is normally injectedintravenously, in order to be directly available in the blood stream.However, the present invention shows that the compositions providedherein are surprisingly more effectively absorbed into the bloodstreamafter SC administration. To be able to administer FVIIa subcutaneouslyserves as an advantage as it can be used for prophylactic applications.Subcutaneous injections are also much easier for patients toself-inject, and are advantage when the patients are very young andtheir veins are small and difficult to find.

Hence, the subcutaneous application can be used for prophylactictreatment.

Example 9 Comparative Pk-Pd Study of Recombinant Mod-5014 Vs. NovoSeven®Following Subcutaneous Administration in SD Rats Study Objectives

To determine the pharmacokinetic and pharmacodynamic parameters ofMOD-5014 versus commercial rFVIIa in SD rats following a single SCadministration.

To compare two independent experiments (05010 & 05034) containingMOD-5014 products originated from two different clones (clone no. 28 vs.61) by their pharmacokinetics parameters.

Experimental Methods

Animals

24 males SD rats arrived from Harlan Laboratories Israel, Ltd, at least4 days before the injections begin. The animals were healthy youngadults, at ˜200 gr at study initiation. The body weight variation ofanimals at the time of treatment initiation should not exceed ±20% ofthe mean weight of each sex. The health status of the animals used inthis study is examined on arrival. Only animals in good health areacclimatized to laboratory conditions and are used in the study.

Clotting assay of FVIIa—STACLOT VIIa-Rtf

The recombinant soluble tissue factor (rsTF) used in this assay isutilizing the FVIIa specificity to construct a FVIIa clotting test.rsTF, In the presence of FVIIa, calcium and phospholipids producecoagulation of plasma, without activating FVII to FVIIa.

The observed clotting time in this system has an inverse relationshipwith the FVIIa content in the tested sample, with no interference ofFVII presence in the sample.

FVIIa activity was evaluated for both NovoSeven® followingreconstitution and MOD-5014 prior to each study. FVIIa specific activitywas calculated based on A280. When comparing specific activity of thetwo molecules, which differ in MW, compensation must be made in order tonormalize the activity (i.e. because of the molecular weight difference,the number of active sites in 1 mg of NovoSeven® is 1.185 fold higherthan in MOD-5014).

PK Solver Software

The pharmacokinetic parameters were calculated using PK solver software.The IV administration curve analyzed as two compartmental CA bolus, andthe SC administration as NCA Extravascular—Log linear trapezoidalanalysis. Half-life, AUC, clearance and volume distributionspecifications were calculated and the output parameters were studied incomparison between groups of experiments.

Experimental Materials

Experiment no. 05010:

-   -   C. NovoSeven® RT: (Lot # AU61553 prepared on 31.7.12*) FVIIa        concentration by A280: 0.86 mg/ml. FVIIa Staclot activity assay:        56,867 U/mg. Injected dose: 946 μg/kg. *Pool of NovoSeven®        aliquots, all from the same Lot no.    -   D. Clone 28: MOD-5014 RS12-001: 0.77 mg/ml** based on A280.        FVIIa Staclot activity assay: 34,162 U/mg. Injected dose: 850 μg        FVIIa/kg.

Experiment no. 05034:

-   -   E. NovoSeven® RT: (Lot #AU61347 prepared on 1.1.13) FVIIa        concentration by A280: 0.82 mg/ml, diluted to 0.4 mg/ml with        sterile NS buffer. FVIIa Staclot activity assay: 55,688 U/mg.        Injected dose: 360 μg/kg and 20,047.7 U/kg.    -   F. Clone 61: MOD-5014 Batch 75: 1.9 mg/ml** based on A280,        diluted to 0.89 mg/ml with formulation buffer. Injected dose:        20,047.7 U/kg. FVIIa clotting activity: 25,002* U/mg based on        FVIIa Staclot activity assay.    -   G. Clone 61: MOD-5014 Batch 81A: 2.36 mg/ml based on A280        (filtered on the morning of study day and re-measured at 280        nm), diluted to 0.4 mg/ml with formulation buffer. Injected        dose: 360 μgFVIIa/kg. FVIIa clotting activity: 24943 U/mg based        on FVIIa Staclot activity assay.    -   H. Clone 61: MOD-5014 Batch 81A: 2.36 mg/ml based on A280,        diluted to 0.89 mg/ml with formulation buffer. Injected dose:        20,047.7 U/kg. FVIIa clotting activity: 24,943 U/mg based on        FVIIa Staclot activity assay.

Study Outlines

Experiment no. 05010

MOD-5014 and NovoSeven® were administered in a single intravenous orsubcutaneous injection to SD Rats in a dose of 0.9 mg/kg body weight.Blood samples were drawn from sinus orbital eye from 3 rats alternatelyat 0.5, 4, 8, 12, 24, 34, 48 and 58 hours post dosing. Citrated plasma(0.32%) was prepared immediately after sampling and stored at −20° c.until analysis. The study was performed at “Science in Action,”Nes-Ziona. FVIIa clotting activity level was evaluated and detailed PKanalysis was performed at Prolor-Biotech.

TABLE 57 Study design 05010 No. of animals/ Dose Time- No. of group/Level Injected Points Treated Test animals/ Time Dose (μg/ Vol. hoursGroups Article group point Route Gender kg) (μl) post-dose) A rFVIIa 6 3IV M 946 220 0, 0.5, 4, 8, (NovoSeven® ) 12, 24, 34, 48, 58 B rFVIIa 6 3IV M 850 220 0, 0.5, 4, 8, RS12-001 12, 24, 34, (clone 28) 48, 58 CrFVIIa 6 3 SC M 946 220 0, 0.5, 4, 8, (NovoSeven® ) 12, 24, 34, 48, 58 DrFVIIa 6 3 SC M 850 220 0, 0.5, 4, 8, RS12-001 12, 24, 34, (clone 28)48, 58

Experiment no. 05034

MOD-5014 and NovoSeven® were administered in a single subcutaneousinjection to SD Rats in a dose of 0.9 mg/kg body weight. Blood sampleswere drawn from sinus orbital eye from 3 rats alternately at 0.5, 2, 4,6, 8, 12, 24, 34, 48 and 72 hours post dosing. Citrated plasma (0.32%)was prepared immediately after sampling and stored at −20° C. untilanalysis. The study was performed at “Science in Action,” Nes-Ziona.

FVIIa clotting activity level was evaluated and detailed PK analysis wasperformed at Prolor-Biotech.

TABLE 58 Study design 05034 No. of Dose Dose animals/ Level Level Time-group/ per per Injected Points Treated. Test Time- Dose Animal AnimalVol. hours Groups Article point*** Route Gender (μg/kg) (U/kg) (μl)post-dose) A FVIIa 3 SC M 360 20047.7 207 0, 0.5, 2, 4, (Novo- 6, 8, 12,24, Seven ®) 34, 48, 72 B FVIIa 3 SC M 801.84 20047.7 207 0, 0.5, 2, 4,75 6, 8, 12, 24, (clone 34, 48, 72 61) C FVIIa 3 SC M 360 8979.48 207 0,0.5, 2, 4, 81A 6, 8, 12, 24, (clone 34, 48, 72 61) D FVIIa 3 SC M 803.7420047.7 207 0, 0.5, 2, 4, 81A 6, 8, 12, 24, (clone 34, 48, 72 61)

Results

FVIIa activity in blood samples was quantitated using STACLOT VIIa-rTFkit (Stago). Pharmacokinetic profile was calculated for each protein andis the mean of 3 animals at each time point.

Experiment no. 05010

FIG. 35 presents the PK profile of FVIIa following IV and SCadministration of either NovoSeven® or MOD-5014. Summary of FVIIaactivity values for each time point is presented in Table 59. IV and SCadministration have different PK pattern as presented in FIG. 35 similarto previous results. The Cmax following IV injection is higher than thatobtained after SC injection, due to the presence of the drug immediatelyfollowing administration in the blood (measured at 0.5 hr, Table 59 andTable 60). However, after SC administration drug molecules transfer tointracellular matrix and tissues, thus Cmax can be measured only after 2hr from injection. The total recovery of the drug after SCadministration is lower than Cmax value after IV injection.

8 hr after injection, Novoseven® manifested an equal PK pattern wheninjected by either IV or SC, (FIG. 35). Moreover, clotting activity forthe NovoSeven®-treated mice was undetectable at time points later than12 hours, while MOD-5014-treated mice continued to retain measurableactivity at 58 hours post dosing (Table 59 and FIG. 35).

TABLE 59 FVIIa clotting activity of MOD-5014 vs. NovoSeven ® followingIV or SC administration Time NovoSeven ® IV (A) MOD-5014 IV (B)NovoSeven ® SC (C) MOD-5014 SC (D) (hr) mU/ml % CV mU/ml % CV mU/ml % CVmU/ml % CV  0.5 304651.7 18.7 232818.3 5.0 11491.7 2.4 3 691.7 19.0  440068.3 7.8 62085.0 9.5 21385.0 22.6 12018.3 15.8  8 5276.7 2.5 25931.76.1 5525.0 32.5 6445.0 2.2 12 255.0 13.8 5633.3 9.3 297.7 41.4 924.724.1 24 1.3 7.1 251.3 11.8 1.3 89.2 249.3 60.3 34 0.0 78.3 4.5 0.0 63.785.5 48 29.0 9.9 0.0 35.0 47.2 58 10.3 4.6 0.0 13.7 33.5 Afterbackground reduction: 15 mU/ml.

TABLE 60 PK parameters of MOD-5014 vs. NovoSeven ® following IV or SCadministration C. IV MOD- PK Parameters Novoseven RT (A) 5014 (RS12-001) (B) Half-life-α (0.5-4 hr) 0.24 1.04 Half-life-β (4-58 hr) 1.313.17 AUC o-inf mU/ml * h 702467.95 820778.67 Vss [U/Kg/(mU/ml)] 0.130.13 CL [(U/Kg)/(mU/ml)/h] 0.08 0.04 MRT (hr) 1.74 3.62 D. SC MOD- PKParameters Novoseven RT (B) 5014 (RS 12-001) (C) Half-Life (hr) 1.407.78 Cmax (mU/ml) 21385.00 12018.33 AUC 0-inf (mU/ml * h) 115099.7284158.87 MRT 0-inf (hr) 4.32 7.04 Vz/F (U/Kg)/(mU/ml) 0.95 3.88 Cl/F(U/Kg)/(mU/ml)/h 0.47 0.35

Experiment no. 05034

FIG. 36 presents the PK profile of FVII a following SC administration ofeither NovoSeven® or MOD-5017. Two different batches of clone no. 61(#75 and #81) were examined in the same concentration or the sameactivity units, compared to NovoSeven®. Summary of FVIIa activity valuesfor each time point is presented in Table 61.

The results indicate a similar PK pattern after SC administrationcorresponding to previous experiments. Moreover, clotting activity forthe NovoSeven® treated mice was undetectable at time points later than12 hours, while MOD-5014 treated mice continued to retain measurableactivity at 24 hours post dosing (Table 61 and FIG. 36; and afterbackground reduction: 56 mU/ml (8, 12 hr) or 32 mU/ml (0.5, 2, 6, 14hr)).

Clone no. 61 batch #81 (D) Cmax (1,301 mU/ml) was lower than the Cmaxvalues of clone no. 61 batch #75 (B) and NovoSeven® (A) (3,521 mU/ml and5,908 mU/ml respectively), although they were all injected by the sameunit activity (Table 61). However, batch #75 (B) and #81 (D) have thesame activity units (559 mU/ml and 478 mU/ml respectively) measured 8 hrafter injection (Table 61 and Table 62; and after background reduction:56 mU/ml (8, 12 hr) or 32 mU/ml (0.5, 2, 6, 14 hr)).

TABLE 61 FVIIa clotting activity of MOD-5014 (Clone 61 #75, #81) vs.NovoSeven ® following single SC administration. MOD-5014 MOD-5014MOD-5014 Clone 61 Clone 61 Clone 61 Batch Batch 81A Batch NovoSeven 75(B)- (C)-equal 81A (D)- ® (A) equal U/kg conc. FVIIa μg/kg equal U/kgTime % % % % (hr) mU/ml CV mU/ml CV mU/ml CV mU/ml CV  0.5 3271.3 46.5350.3 26.6 101.3 24.1 208.7 51.2  2 5908.0 18.1 3521.3 70.9 1294.7  7.01301.3 31.6  6 1411.7 23.6 1349.7 45.6 425.3 27.6 663.0 13.4  8 1029.012.4 559.3 52.7 152.7 19.5 478.0 25.4 12 121.3  9.9 563.0 17.4 148.736.3 712.7 16.2 24 1.0 25.0 117.0 41.9 21.3 36.4 99.0 36.7 Afterbackground reduction: 56 mU/ml (8, 12 hr) or 32 mU/ml (0.5, 2, 6, 14 hr)

TABLE 62 PK parameters of MOD-5014 (Clone 61 #75, #81) vs. NovoSeven ®following single SC administration. MOD-5014 MOD-5014 Clone 61 Clone 61Batch MOD-5014 Clone 61 Batch 81A PK NovoSeven ® 75 (B) - equal Batch81A (C) - equal (D) - equal Parameters RT (A) U/kg conc. FVIIa μg/kgU/kg Half-Life (hr) 1.67 5.70 4.62 6.41 Cmax (mU/ml) 5908.00 3521.331294.67 1301.33 AUC 0-inf 24688.18 20456.96 6260.23 13098.16 (mU/ml * h)MRT 0-inf (hr) 3.73 7.86 6.40 10.59 Vz/F 1.96 8.06 9.55 14.15(U/Kg)/(mU/ml) Cl/F 0.81 0.98 1.43 1.53 (U/Kg)/(mU/ml)/h

This report summarized two PK studies; 05010 & 05034. The resultsprovide specific insight on the impact of CTP fusion to FVII on proteinhalf-life and clearance in subcutaneous administration and address theparadigm of its specific activity following this modification. In thesestudies, SD rats were administered with a single SC injection ofMOD-5014 originated from two clones, and two different batches, comparedto recombinant commercial FVIIa (NovoSeven®). The components wereinjected at similar FVIIa concentration (μg/Kg) or at the same activitylevel (U/Kg) and the PK activity based analysis was performed.

The purpose of the first study was to verify the different PK parametersafter IV and SC administration. Based on this study we can conclude thatthere is a difference between the PK pattern measured after IV or SCadministration. A t^(1/2) of 7.78 hr measured after MOD-5014 SCinjection, and only 4.2 hr after IV injection. AUC values were the sameTable 60.

The second study however, focused on the differences between two batchesof MOD-5014 clone no. 61, which were injected by the same FVIIaconcentration or at an equal activity unit, compared to NovoSeven®. Inthis study we showed that clone 61 batch #75 manifested better PKparameters than batch #81. Batch #81, which was injected by the sameunit activity level, had lower Cmax from an unknown reason. Moreover,the same Cmax was measured when injecting clone 61 batch #81 in twodifferent doses (by FVIIa concentration or by unit activity), instead of2.5 fold between the two activity values. Following analysis of bothstudies together, we can conclude that clone 28 manifested a prolongedt^(1/2) parameter that clone 61 #75 (the better batch) after SCinjection (7.78 hr and 5.7 hr respectively, Table 62). The results showthat dissimilar time point samples create different PK pattern, whichlead to variation in the PK curves. The patterns of the curves can teachus more about the drug behavior in the blood. Therefore, we decided todetermine the time points similar to those detected by Baxter (0, 0.5,2, 6, 8, 12, 24, 34, 48, 72 hr). Moreover, the FVIIa concentration in05010 experiment was too high, and was revised in the following SCexperiment (05034). For future PK studies, we decided to inject thecomponent at 360 μg FVIIa/kg for a dose.

Example 10 Warfarin Treated Rats as a Model for Evaluating Factor VIIain vivo Materials & Methods

PT Assessment: SD rats were given orally 10 mg/Kg of Warfarin and at adesignated time point plasma was collected and prothrombin time (PT) wasmeasured using a standard procedure. In order to assess the long termhemostatic effect Placebo, NovoSeven® or MOD-5014 were injected to theWarfarin treated animals and PT was measured.

Tail Clip Challenge: Warfarin treated animals were injected withPlacebo, NovoSeven® or MOD-5014 at designated time points the animalswere challenged by complete cut of the tail tip (0.5 cm from the tip)and bleeding intensity was measured in gr for 30 min post transection.

Results

Warfarin Administration to SD-Rats Results in a Prolongation of PT andaPTT.

Warfarin prevent the reduction of vitamin K, and consequently decreasesvitamin K dependent coagulation factors concentration in the blood. MaleSD rats received oral treatment of warfarin. The reduction of Vitamin Kdependent coagulation factors was accompanied by prolongation of PT andaPTT. Results are presented in FIG. 37.

Due to coagulation-factors wash out from the blood, PT and aPTT valuesincrease gradually in the first 48 hours following warfarinadministration. The effect decreases after that.

Warfarin Effect can be Restored by Acute IV Treatment with NovoSeven® orMOD-5014.

SD-rats received a pre-treatment of Warfarin. 24 hours later, MOD-5014,NovoSeven® or buffer were injected intravenous blood samples were drawn15 minutes post injection. 15 min post injection, MOD-5014 as well asNovoSeven® successfully restored PT values to normal (FIG. 38). Theeffect of increasing dose of MOD-5014 and NovoSeven® on PT values inwarfarin treated rats.

SD-rats were treated with 10 mg/Kg warfarin in parallel to 100-1000μg/Kg MOD-5014 or NoveSeven IV injection. 24 hours post treatment, PTwas determined in plasma samples. NovoSeven® injected 24 hours before PTdetermination, did not have any significant effect on PT values in allthe doses tested. In contrast, MOD-5014 shows a dose-response behavior24 hours after administration (FIG. 39).

SD-rats were treated with 10 mg/Kg warfarin in parallel to 1000 μg/KgMOD-5014 or NoveSeven IV injection. PT was determined in plasma samples10, 24, 36 and 48 hours post treatment. MOD-5014 restored PT values tonormal up to 48 hours post dosing, while the effect of NovoSeven® nolonger exists after 24 hours (FIG. 40).

MOD-5014's Long Lasting Effect can be Demonstrated by Tail Clip Assay inWarfarin Injected Rats

SD-rats were treated with Warfarin 24 hours before tail clip. Rats wereanesthetized and placed on a warm pad, the tail tip was placed in 37° C.saline and a complete amputation of the tail was performed 0.5 cm fromtail tip. Blood was collected for 30 minutes and blood loss wasdetermined by weight.

Vehicle or 500 μg/Kg MOD-5014 or NovoSeven® was administrated 15 min, 24or 48 hours before tail clip. Results are presented in FIG. 41. Ratstreated with warfarin lost 5 fold more blood than naïve rats. 15 minpost injection, tail clip of MOD-5014 and Novoseven treated ratsresulted in reduced bleeding which is comparable to naïve rats. Theeffect of MOD-5014 is completely preserved 24 hours post injection, andpartially preserved after 48 hours.

Sub-Cutaneous Injection of MOD-5014 is Also Demonstrating a Long LastingEffect.

SD-rats were treated with 10 mg/Kg warfarin in parallel to 2000 μg/KgMOD-5014 or NoveSeven SC injection. PT was determined in plasma samples10, 24, 36 and 48 hours post treatment.

MOD-5014 is able to restore PT values to normal up to 48 hours postdosing, while the effect of NovoSeven® no longer exists after 24 hours(FIG. 42).

SC Injection of MOD-5014 Reduces Blood Loss for at 48 Hours.

SD-rats were treated with Warfarin 24 hours before tail clip. Rats wereanesthetized and placed on a warm pad, the tail tip was placed in 37° C.saline and a complete amputation of the tail was performed 0.5 cm fromtail t

ip. Blood was collected for 30 minutes and blood loss was determined byweight.

Vehicle or 1000 μg/Kg MOD-5014 or NovoSeven® was SC administrated 15min, 24 or 48 hours before tail clip. Results are presented in FIG. 43.

Example 11 Comperative Assessment of Clotting Activity of MOD-5014 andNovoseven®

Study Objectives—(I) To characterize the in vitro clotting activity andFX activation under different conditions of MOD-5014 in comparison withNovoSeven®. (II) To compare ex vivo, prothrombin time (PT) and activatedpartial thromboplastic time (aPTT) profiles in human hemophilia andFVII-deficient plasma upon MOD-5014 and NovoSeven® spiking.

Materials and Methods

Materials—MOD-5014 GMP-1: 2.5 mg/ml (based on A280) and NovoSeven® Lot#CU60430: 0.943 mg/ml (based on A280)

Method—Clotting Assay

The clotting activity of FVIIa was measured using commercially availableStaclot VIIa-rTF kit (Ref#00281, Stago). This method included clottingtime measurement of FVII-deficient plasma using the STA Compact MAX orStart4 instruments. Specific amounts of FVIIa were added to the plasmafollowing the addition of phospholipids, Ca²⁺, and recombinant solubletissue factor (rsTF). The latter is the extracellular portion of thenative tissue factor, which can no longer activate FVII to FVIIa by autoactivation. However, it possesses a cofactor function specific forfactor VIIa. The FVIIa bound to soluble tissue factor converts factor Xto the active factor Xa. The observed clotting time has an inverserelationship with FVIIa level in the plasma, since the soluble tissuefactor does not activate FVII to FVIIa. The obtained clotting time wasconverted to activity (mU/ml) using a FVIIa standard curve and thespecific activity was calculated based on FVIIa protein concentration.This method provided the potential in vitro activity of FVIIa, with thelimitation of using sTF, which only partially mimics the in vivosetting.

Method—FVII Chromogenic Assay

MOD-5014 and NovoSeven® potency were assessed by the commerciallyavailable kit BIOPHEN FVII (Ref#221304, HYPHEN BioMed). This is achromogenic assay intended for testing FVII activity. FVII forms anenzymatic complex with tissue factor and converts factor X into theactivated factor Xa in the presence of phospholipids and calcium. FactorX is present in the assay in a constant concentration and in excess. Theconcentration of activated factor Xa is measured by its activity on aspecific chromogenic substrate (SXa-11), which it cleaves to generatepNA. The amount of pNA is directly proportional to Factor X activity,and there is a direct relationship between the amount of Factor VII andthe level of Factor Xa activity, measured by the amount of pNA releasedand determined by color development at 405 nm.

Method—Prothrombin Time (PT) and Activated Partial Thromboplastic Time(aPTT)

Prothrombin Time (PT) and Activated Partial Prothrombin Time (aPTT) weremeasured using a Siemens CA-1500 autoanalyzer and validated usingroutine clinical human plasma diagnostic testing at A.M.L.

MOD-5014 GMP-1:

2.5 mg/ml based on absorption at A280, diluted to 0.5-0.0008 mg/ml withhemophilic human plasma/FVII deficient plasma.

FVIIa Clotting Activity:

16,720 U/mg based on FVIIa Staclot activity assay.

NovoSeven® Lot#CU60430:

0.943 mg/ml based on absorption at A280, diluted to 0.5-0.0008 mg/mlwith hemophilic human plasma/FVII deficient plasma.

FVIIa Clotting Activity:

50,494 U/mg based on FVIIa Staclot activity assay.

Matrix:

Human plasma (FVIII deficient) BIORECLAMATION (Cat# HMPLCIT-FACT8DEF,Lot# BRH779222-BRH779233). FVII Deficient plasma, Cat#HBM-DP030K.

Results Potency Determination

The specific activities of MOD-5014 and NovoSeven® were evaluated usinga qualified Staclot VIIa-rsTF kit. The average specific activityobtained for MOD-5014 (batches RS005, GMP1, ER01) and NovoSeven® asdetermined by 4 independent assays are presented in Table 63 below.

TABLE 63 Specific activities of MOD-5014 and NovoSeven ® Run Run Run RunAvg % #1 #2 #3 #4 (U/mg) CV MOD-5014 17041 14223 16344 14644 15563 8.6batch ER01 MOD-5014 15894 17390 17819 15775 16720 6.2 batch GMP1MOD-5014 23300 20471 17790 18323 19971 12.5 batch RS005 NovoSeven ®46889 45900 49522 47644 47489 3.2

Conclusion:

MOD-5014 specific activity was 2 to 2.5-fold lower than NovoSeven®. Thismight be a consequence of reduced molar content of FVIIa in MOD-5014when spiking on mass base rather than on molar base, as MOD-5014consists of 83.4% FVIIa with 3 CTP cassettes attached at the C-terminus.

Clotting Activity Inhibition in the Presence of TFPI:

The clotting activity of MOD-5014 and NovoSeven® was evaluated in thepresence of tissue factor pathway inhibitor (TFPI), a natural inhibitorif FVIIa. The inhibitor was added at a range of concentrations (3125ng/ml to 0.006 ng/ml) following the addition of MOD-5014 or NovoSeven®at a fixed concentration, FVII-deficient plasma, tissue factors andphospholipids, and was incubated 15 min at 37° C. The observed specificactivity was converted to inhibition %. The assay was repeated 3 timesand the mean results are presented in Table 64 and FIG. 44.

TABLE 64 Mean Activity Inhibition in the Presence of TFPI NovoSevenMOD-5014 TFPI conc. TFPI Activity % Activity % (ng/mL) log conc. (mU/mL)Inhibition (mU/mL) Inhibition 0 NA 72.3 NA 66.8 NA 1250 3 2.9 96 7.3 89625 3 9.5 87 13.2 80 62.5 2 45 38 47.4 29 6.25 1 55.3 24 56.7 15 0.625 054.5 25 56.7 15

Conclusion:

TFPI inhibited NovoSeven® and MOD-5014 at a similar dose-dependentmanner. The difference in the values might be a consequence of assayvariability, as reported in method qualification (% CV<25%).

Clotting Activity Inhibition in the Presence of Heparin:

The clotting activity of MOD-5014 and NovoSeven® was measured in thepresence of heparin at a wide range of concentrations. Heparin was addedfollowing the addition of a fixed concentration of MOD-5014 orNovoSeven®, FVII-deficient plasma, tissue factors and phospholipids, andwas incubated 15 min at 37° C. The results are presented in Table 65.

TABLE 65 Activity Inhibition in the Presence of Heparin NovoSevenMOD-5014 Heparin Conc. Activity Activity % (U/μl) (mU/ml) % Inhibition(mU/ml) Inhibition 0 68.8 NA 56.9 NA 1 0 100 0 0 0.5 0 100 0 0 0.25 0100 0 0 0.1 2.1  97 2.1 96 

Conclusion:

Heparin possesses high potency in this specific assay, as over 96%inhibition was observed for both MOD-5014 and NovoSeven® even at anextremely low concentration (0.1 U/μl).

Clotting Activity in the Presence of Anti-Thrombin (AT):

The specific activities of MOD-5014 and NovoSeven® were evaluated in thepresence of Anti thrombin III (ATIII), which is a mild inhibitor ofFVIIa. Anti-thrombin was added at a range of concentrations (525 μg/mlto 0.01 ng/ml) following the addition of MOD-5014 or NovoSeven®,FVII-deficient plasma, tissue factors and phospholipids, and wasincubated 15 min at 37° C. The observed specific activity was convertedto inhibition %. The results are presented in Table 66 and FIG. 45.

TABLE 66 Activity Inhibition in the Presence of AT III NovoSevenMOD-5014 AT conc. AT log Activity Activity % (ng/mL) conc. (mU/mL) %Inhibition (mU/mL) Inhibition 0 NA 88.1 NA 70.8 NA 525000 6 0 100  0100  105000 5 30.6 65 27 62 10,500 4 61.5 30 53 25 1,050 3 66.8 24 53.425 105 2 70.2 20 53.3 25 10.5 1 68.4 22 50.7 28 1.050 0 69.5 21 52.9 25

Conclusion:

MOD-5014 and NovoSeven® were inhibited at a similar manner by AT III.

Factor X Activation by NovoSeven® and MOD-5014:

The potency of MOD-5014 and NovoSeven® was evaluated and the averageEC₅₀ values obtained for MOD-5014 and NovoSeven® were calculated (0.41and 0.38 ng/ml, respectively). The results are presented in Table 67 anda representative dose-response curve is presented in FIG. 46.

TABLE 67 FX activation by MOD-5014 and NovoSeven rFVIIa O.D Conc. LogMOD- (ng/ml) Conc. 5014 NovoSeven 100.00 2.00 0.385 0.363 20.00 1.300.358 0.365 4.00 0.60 0.368 0.352 0.80 −0.10 0.286 0.303 0.160 −0.800.17 0.178 0.0320 −1.49 0.127 0.138 0.00640 −2.19 0.117 0.133 0.00128−2.89 0.111 0.123

FX Activation in the Presence of TFPI:

The potency of MOD-5014 and NovoSeven® was evaluated in the presence ofTFPI. The latter was added at a range of concentrations (20 μg/ml to0.002 ng/ml) to two concentrations of MOD-5014 and NovoSeven® (EC₇₀)(0.6 and 4 ng/ml) and FX activation was measured. The results arepresented in Table 68 and FIG. 47. The assay performed in the presenceof NovoSeven® and MOD-5014 at a concentration of 4 ng/ml is presented inTable 69 and FIG. 48.

TABLE 68 Factor X Activation in the Presence of TFPI (#1) O.D TFPI Conc.MOD-5014 NovoSeven ® (ng/ml) Log Conc. (0.69 ng/ml) (0.64 ng/ml)20000.00     4.30 0.346 0.429 4000.00     3.60 0.691 0.83 800.00    2.901.006 1.044 160.00    2.20 1.11 1.337 32.00    1.51 1.36 1.384 6.40  0.81 1.384 1.418 1.28   0.11 1.387 1.466 0.2560  −0.59 1.457 1.4280.051200 −1.29 1.454 1.518 0.010240 −1.99 1.446 1.514 0.002048 −2.691.478 1.504 Control 1.51 1.51

TABLE 69 Factor X Activation in the Presence of TFPI (#2) TFPI Conc. LogNovoSeven ® MOD- (ng/ml) Conc. (4 ng/ml) 5014 (4 ng/ml) 20000.00    4.30 0.206 0.17 4000.00     3.60 0.406 0.38 800.00    2.90 1.016 0.836160.00    2.20 1.338 1.143 32.00    1.51 1.496 1.465 6.40   0.81 1.5881.541 1.28   0.11 1.661 1.565 0.2560  −0.59 1.697 1.616 0.051200 −1.291.726 1.604 0.010240 −1.99 1.703 1.638 0.002048 −2.69 1.715 1.653Control 0 1.656 1.581

Conclusion:

MOD-5014 and NovoSeven® demonstrated a very similar inhibition curve ofFX activation in the presence of TFPI at both concentrations of thecompound.

FX Activation in the Presence of TFPI and Heparin—

The potency of MOD-5014 and NovoSeven® was evaluated in the presence ofTFPI and heparin. TFPI at different concentrations (20 μg/ml to 0.002ng/ml) and 1 U/μl heparin were added to a constant concentration ofMOD-5014 and NovoSeven® (0.7 ng/ml) and FX activation was measured. Theresults are presented in Table 70 and FIG. 49.

TABLE 70 FX Activation in the Presence of TFPI and Heparin NovoSeven ®MOD- TFPI (0.7 ng/ml) + 5014 (0.7 ng/ml) + Conc. Log 1 U/μl 1 U/μl(ng/ml) Conc. heparin heparin 20000.00     4.30 0.222 0.15 4000.00    3.60 0.241 0.115 800.00    2.90 0.409 0.32 160.00    2.20 0.764 0.53932.00    1.51 1.014 0.915 6.40   0.81 1.207 1.164 1.28   0.11 1.3091.261 0.051200 −1.29 1.373 1.347 0.010240 −1.99 1.305 1.293 0.002048−2.69 1.335 1.266 Control 0 1.473 1.373

Conclusion:

MOD-5014 and NovoSeven® exhibited similar activation of FX in thepresence of TFPI. Heparin had no significant influence on the inhibitionprofile when added with TFPI.

FX Activation in the Presence of Anti-Thrombin:

The potency of MOD-5014 and NovoSeven® was evaluated in the presence ofanti-thrombin (AT III). Different concentrations of AT III (1.68 mg/mlto 0.16 μg/ml) were added to a constant concentration of MOD-5014 andNovoSeven® (0.7 ng/ml) and FX activation was measured. The results arepresented in Table 71 and FIG. 50.

TABLE 71 FX Activation in the Presence of Anti-Thrombin AT O.D conc. LogMOD- (μg/ml) conc. NovoSeven 5014 1680  3.2 0.784 0.656 500 2.7 0.9330.975 100 2.0 1.138 0.974  20 1.3 1.151 1.112  4 0.6 1.172 1.135    0.8−0.1 1.2 1.075    0.16 −0.8 1.234 1.182 control 1.283 1.09

Conclusion:

MOD-5014 and NovoSeven® exhibited similar activation of FX at thepresence of AT III.

FX Activation in the Presence of Constant AT III Concentration andVarying Heparin Concentrations:

Anti-thrombin III (AT III) was diluted to a constant concentration (20μg/ml) and was added to a constant concentration of MOD-5014 andNovoSeven® (0.7 ng/ml). Heparin was added at different concentrations(6.25 U/μl-0.002 U/μl) to the mixture and FX activation was measured.The results are presented in Table 71 and FIG. 51.

TABLE 71 FX Activation in the Presence of Heparin at DifferentConcentrations Heparin conc. Log O.D. (U/μl) conc. NovoSeven MOD-50146.25 0.8 1.002 1.024 1.25 0.1 0.98 0.896 0.25 −0.6 1.039 0.917 0.01 −2.01.198 1.139 0.002 −2.7 1.242 1.294

Conclusion:

MOD-5014 and NovoSeven® exhibited similar and moderate inhibition byheparin at constant AT III concentrations.

PT and aPTT Measurements

PT and aPTT measurements are presented in Table 72.

TABLE 72 PT (sec) aPTT (sec) Tested In In FVII In In FVII Testconcentration Required Hemophilic Deficient Hemophilic Deficient article(mg/ml) dilution Plasma Plasma Plasma Plasma MOD-5014 0.5 5 10.0 10.221.0 21.0 GMP-1 0.1 25 8.7 8.9 22.8 21.0 2.5 mg/ml 0.02 125 8.6 8.8 30.9<21.0 0.004 625 8.5 8.8 45.0 24.0 0.0008 3125 8.8 9.3 62.5 25.9Novoseven 0.5 1.9 No coagulation No coagulation 0.943 mg/ml 0.1 9.3 8.58.7 21.0 21.0 0.02 47 8.3 8.6 26.6 21.0 0.004 234 8.3 8.6 38.9 22.70.0008 1169 8.5 8.7 55.0 25.4 Control 0 0 11.9 No coagulation 87.1 27.5(Only Plasma) Normal values 11-13.5 25-35 Extrinsic pathway Intrinsicpathway

Conclusion:

The PT and aPTT measurements were comparable for both MOD-5014 andNovoSeven® when spiked at a similar range of concentrations.

SUMMARY

MOD-5014 activity was evaluated by a variety of in vitro and ex vivomethods that assessed different aspects of its coagulation activity incomparison to NovoSeven® at equal mass. Initially, MOD-5014 activity wascompared to NovoSeven® in the qualified Staclot FVIIa assay. The studydemonstrated that MOD-5014 activity is 2 to 2.5-fold lower than that ofNovoSeven. A factor X activation test by a chromogenic assay in thepresence of TF, phospholipids and calcium also proposed a slightly lowermeasure of activity of MOD-5014 in comparison to NovoSeven®, asreflected by EC₅₀.

The variability between methods might be due to differences in assaysensitivity or endpoint (soluble vs. full-chain TF and clotting time vs.OD measurement, respectively). The lower MOD-5014 activity might be dueto the fact that 84.4% of MOD-5014 corresponds to FVIIa and 15.6%corresponds to CTP.

MOD-5014 inactivation in the presence of TFPI, a major FXa-dependentinhibitor of the extrinsic coagulation pathway, was evaluated by the twoin vitro assays mentioned above (Staclot and factor X activation).Incubating MOD-5014 or NovoSeven® at two fixed concentrations withincreasing concentrations of TFPI resulted in a dose-dependent reductionin clotting or FXa enzymatic activity. Both compounds demonstrated avery similar de-activation pattern, reflected by clotting inhibition.

Anti-thrombin III was previously reported to inhibit factor VIIa at aslow rate and also demonstrated augmented inhibition at the present ofheparin. Anti-thrombin III demonstrated a similar inhibition pattern ofboth MOD-5014 and NovoSeven® when both compounds were spiked withincreasing concentrations of AT III. This pattern was maintainedfollowing the addition of heparin, producing a pronounced inhibitoryeffect.

Example 12 Comparative In Vitro Assessment of Mod-5014 and Novoseven® inThrombin Generation Andcoagulation Efficiency

Study Objectives—

(I) Comparative assessment of MOD-5014 and NovoSeven® by thrombingeneration (TG) in inhibitory platelets rich plasma with high titer ofinhibitory antibodies at low and high phospholipids concentration, and(II) comparative assessment of MOD-5014 and NovoSeven® bythromboelastography (TEG) in inhibitory platelets rich plasma with hightiter of inhibitory antibodies.

Materials and Methods

Materials—

MOD-5014: 2.0 mg/ml and NovoSeven® 1.0 mg/ml, stored frozen (−60 to −80°C.). No dose formulation preparation was required. Materials were thawedonly once prior to dosing.

Method—Thrombin Generation (TG) in Low and High PhospholipidConcentrations

Human plasma originated from patients with high titer of anti FVIIIinhibitory Abs was spiked with increased concentration of MOD-5014 orNovoSeven®, coagulation was stimulated by relipidtaed rhTissue Factor(TF) and a high concentration of phospholipid micelles (Reagent RChigh)mimicking the in vivo situation.

Method—Thromboelastography (TEG)

Thromboelastography (TEG) is a method of testing the efficiency ofcoagulation in the blood and is especially important in surgery andanesthesiology. The patterns of changes in strength and elasticity inthe clot provide information about how well the blood can performhemostasis (the halting of blood flow), and how well or poorly differentfactors are contributing to clot formation.

Four values that represent clot formation are determined by this test:the R value (or reaction time), the K value, the angle and the MA(maximum amplitude). The R value represents the time until the firstevidence of a clot is detected. The K value is the time from the end ofR until the clot reaches 20 mm and this represents the speed of clotformation. The angle is the tangent of the curve made as the K isreached and offers similar information to K. The MA is a reflection ofclot strength.

Results

Thrombin Generation:

The generation of thrombin is a fundamental part of the clotting cascadeand as such an estimation of how well a particular individual cangenerate thrombin may correlate with either a risk of bleeding orthrombosis. It describes all the phases of thrombin generation process(initiation, amplification and inhibition of thrombin generation as wellas the integral amount of generated thrombin). According to theexperimental system used, thrombin generation may be influenced by mostof the factors playing a role in in vivo blood coagulation.

The kinetic of thrombin generation was monitored by Technoclone TGA(FIG. 53).

A Dose-dependent increase of peak thrombin and decrease of lag phase andpeak time was observed following spiking with MOD-5014 or Novoseven.

In the second experiment MOD-5014 or NovoSeven® were spiked atincreasing concentrations at the present of TF and at the present of lowconcentration of phospholipid micelles (Reagent RClow).

As anticipated a more pronounced repose was observed when spiking thesample with high PL concentration. Both compounds reached maximal TGresponse at low concentration of PL concentration further confirming itsimportance for proper activation of the clotting cascade. The result aspresented in FIG. 53 and FIG. 54 demonstrate that MOD-5014 in-vitrothrombin generation activity is slightly lower compared to NovoSeven® athigh and low PL concentration and are aligned with data obtained in PPPFVIII deficient plasma.

Coagulation Efficiency:

MOD-5014 and NovoSeven® were added to high titer human FVIII inhibitorplatelet reach plasma (PRP), CaCl₂ and rhTF thromboplastin were added totrigger clot formation. The R and angel were evaluated. As observed inFIG. 55, both MOD-5014 and NovoSeven® decreased the clotting time (R)and increased the rate of clot formation (Angel) of high titer FVIIIinhibitor plasma at a similar concentration dependent manner, whileMOD-5014 demonstrated a minor reduction in its clotting capability.These finding further strengthen the results obtained by ROTEM that CTPattachment doesn't interfere with clot formation.

SUMMARY

Based on the TG and TEG results in FVIII high titer plasma it appearsthat MOD-5014 mechanism of TG and clot formation may be similar toNovoSeven® with a slight reduction in it activity. This might be aconsequence of reduced molar content of FVIIa in MOD-5014 when spikingon mass base rather than on molar base as MOD-5014 consists of 83.4%FVIIa with 3 CTP cassettes attached at the C terminus, and thereforewill require slightly higher concentrations of MOD-5014 to maintain invivo hemostatic. Finally it appears that a mechanism of binding tophospholipid may be maintained following the attachment of CTP to FVIIa.

Example 13 Comparative In Vitro Activity of Mod-5014 and Novoseven®

Study Objectives—(I) A comparative assessment of MOD-5014 and NovoSeven®by thrombin generation (TG) in citrated platelet poor plasma (PPP). (II)A comparative assessment of MOD-5014 and NovoSeven® tissue factor (TF)affinity by thrombin generation in citrated PPP. (III) A comparativeassessment of MOD-5014 and NovoSeven® by thromboelastography (ROTEM) inPPP citrated plasma.

Materials and Methods

Materials—

MOD-5014: 2.6 mg/ml and NovoSeven® 2.6 mg/ml, stored frozen (−60 to −80°C.). No dose formulation preparation was required. Materials were thawedto room temperature prior to administration.

Method (Objective (I))—Thrombin Generation (TG) in Low and HighPhospholipid Concentrations

Thrombin generation was measured according to Livnat et al. (2006, J.Thromb Haemost. 4(1):192-200; 2008, Haemophila. 14(4):782-786; and 2011,Thromb Haemost. 105(4): 688-95). Briefly, pooled plasma was spiked withescalating concentration of MOD-5014 or NovoSeven®. PPP-Reagent LOW(containing 104 tissue factor) or MP-reagent (Diagnostica Stago, Lot PPL1203/01 and MPR 1202/01, respectively) were used as working buffers.Both reagents contained 4 μM phospholipids.

The assay was carried out using two approaches as follow: (a)Re-Calcification only; and (b) Low TF level(1 pM).

Twenty μl of working buffer were placed in round-bottom 96-wellmicrotiter plates. Eighty μl of FVIII-deficient plasma with differentconcentrations of NovoSeven® or MOD-5014 were added to the buffer, asdescribed in Table 73.

TABLE 73 *Predicated Tissue NovoSeven ®/MOD- in vivo factor (TF) 5014spiked concentration Phospholipids concentration concentration (μg/ml)(μg/kg) concentration w/wo 0 0 4 μM 1 pM 1.25 40 4 μM 1 pM 2.5 80 4 μM 1pM 5 160 4 μM 1 pM 10 320 4 μM 1 pM 15 640 4 μM 1 pM *Based on Livnat etal., 2008.

TG was initiated by adding 20 μl of fluorogenic substrate/CaCl₂ buffer(FluCa-kit Thrombinoscope-BV, Diagnostica Stago, Lot FLB 1303/01).Fluorescence was measured using an excitation filter at 390 nm and anemission filter at 460 nm and a fluorometer (Fluoroskan Ascent, Labsystem, Helsinki, Finland). Results were displayed as plots and derivedparameters, i.e. lag time, endogenous thrombin potential (ETP) and peakheight, and were calculated using specialized computer software (version3.0.0.29, Thrombinoscope-BV Maastricht, the Netherlands). Each samplewas tested independently twice in duplicates (run 1 and 2). The meanvalue of the duplicates is provided and compared to a thrombin standard(Thrombin calibrator, Diagnostica Stago, Lot TC 1208/01).

Method—(Objective (II))—Thrombin Generation (TG)

Thrombin generation was measured according to Livnat et al. (2006, 2008,2011). Briefly, pooled plasma was spiked with 3 fairly low escalatingconcentration of MOD-5014 or NovoSeven® to enable a more sensitive anddose-dependent response (1.25, 2.5, 5, and 10 μg/ml). MP-reagentcontaining 4 μM phospholipids was used as working buffer. Eachdesignated sample was spiked with escalating concentrations of TF asdescribed in Table 74 below, and TG was assessed.

TABLE 74 NovoSeven ®/ *Predicated MOD-5014 spiked in -vivo Phospho- TFconcentration concentration lipids con- concentration (μg/ml) (μg/kg)centration w/wo 0 0 4 μM 0, 0.5, 1, 2.5, 5 pM 1.25 40 4 μM 0, 0.5, 1,2.5, 5 pM 2.5 80 4 μM 0, 1, 2, 4, 5 pM 0, 0.5, 1, 2.5, 5 pM 5 160 4 μM0, 0.5, 1, 2.5, 5 pM 10 320 4 μM 0, 1, 2, 4, 5 pM 0, 0.5, 1, 2.5, 5 pM*Based on Livnat et al., 2008TG of each test article at the designated concentrations of TF wasassessed by measurement ETP, lag time and height of thrombin peak. Eachsample was tested independently twice in duplicates (run 1 and 2). Themean value of the duplicates is provided.

Method—Objective (III)—Rotation Thromboelastography (ROTEM)

Pooled plasma from severe FVIII-deficient patients were spiked witheither NovoSeven® or MOD-5014 at 4 concentrations as described in Table75 below, and assessed at the following conditions: (a) addition ofKaolin; (b) with low levels of TF; (c) re-classification.

TABLE 75 NovoSeven ®/MOD-5014 *Predicated in-vivo spiked concentrationconcentration (μg/ml) (μg/kg) 0 0 2.5 80 10 320 15 640

ROTEM measurements were conducted with a ROTEM device (Pentapharm,Munich, Germany) using 300 μL of FVIII-deficient plasma placed intocups, with a subsequent addition of 20 mM CaCl₂, (NATEM), allegic acid(contact activation, INTEM), or low concentration of tissue factor(EXTEM reagent diluted 1:1700). ROTEM tests were performed according tothe manufacturer's instructions at 37° C. and were run for a minimum of45 minutes. The following variables were used: clotting time (CT, sec),i.e. the time between the introduction of CaCl₂ and the beginning ofclotting; alpha-angle (α-angle, degrees) reflecting clot propagation;and maximum clot firmness (MCF, m), which reflects clot strength.

Results

Objective (I): Comparative In Vitro Assessment of MOD-5014 andNovoSeven® by Thrombin Generation (TG)

Thrombin generation has been frequently used for the assessment ofhemostatic effect of FVIIa. It was previously shown that FVIIa mediateschanges in thrombin generation (TG) in FVIII inhibitor plasma samples.In plasma samples spiked with recombinant FVIIa (rFVIIa), TG wasimproved in the absence of tissue factor (TF) while the TG potential ofrFVIIa in vitro was increased as a result of added TF. Simple, classicpharmacokinetic assays are not available for FVIIa due to the complexityof its mechanism of action. Since thrombin is the final productgenerated, a TG assay could be used for the assessment of thepharmacokinetics and potential efficacy of MOD-5014 and NovoSeven®. Thisassay is suitable for monitoring the pharmacokinetics ofinhibitor-bypassing agents during treatment and may be useful forpredicting responses to treatment. Therefore real-time measurement ofthrombin concentration generated in plasma gives valuable informationregarding the homeostasis of the coagulation system.

The objective of this study was to compare the in vitro thrombingeneration ability of MOD-5014 and NovoSeven® in severe hemophilia Aplasma at a range of concentrations that potentially correlate to theproposed clinical doses in the first-in-human (F1H) study. This couldprovide a prediction of the minimal effective dose as part of thepreparations for first in human (F1H) study.

Comparative Thrombin Generation following re-calcification

MOD-5014 and NovoSeven® were spiked at a wide range of concentrations tosevere hemophilia A pooled plasma. The study was repeated twice, and theresults are provided in Tables 76-79 below, and FIGS. 56, 57, 58(A-E),59(A-D), 60, 61, 62(A-E) and 63(A-D).

TABLE 76 (Run #1) NovoSeven ® Derivative Values for FIG. 56 1.25 μg/ml2.5 μg/ml 5 μg/ml 10 μg/ml 15 μg/ml Lag time 29.5 20.67 15.83 12.33 11.5Lag time SD 2.17 0 0.5 0.33 0.17 ETP 141 242 460 727 659.5 ETPSD 13 0115 54 15.5 Peak 6.2 11.45 20.33 33.45 39.85 Peak SD 0.66 0 2.32 0.031.34 tt Peak 44.17 33.67 28.5 22.83 21.33 tt Peak SD 1.83 0 0.5 0.5 0Start Tail 76 76 73 71.5 74 Start Tail SD 0 0 2 3.5 0 Edited 1 1 0 0 1

TABLE 77 (Run #1) MOD-5014 Derivative Values for FIG. 57 Lag time 37.3329.33 20.17 16.17 13.67 Lag time SD 0.67 0.33 0.5 0.5 0 ETP 108.5 144.5295 426 623.5 ETPSD 2.5 0.5 39 19 179.5 Peak 4.85 6.85 12.32 20.32 26.57Peak SD 0.15 0.11 0.34 0.76 2.46 tt Peak 52.5 42.83 34 28 25.33 tt PeakSD 0.83 0.5 0.33 0.67 1 Start Tail 76 76 75 72.5 76 Start Tail SD 0 0 00.5 0 Edited 1 1 1 0 1

TABLE 78 (Run #2) NovoSeven ® Derivative Values for FIG. 60 1.25 μg/ml2.5 μg/ml 5 μg/ml 10 μg/ml 15 μg/ml Lag time 18.7 13.85 12.35 10.35 8.68Lag time SD 0 0.5 0.67 0.33 0.33 ETP 226 269 363.5 554.5 756 ETPSD 0 1626.5 76.5 31 Peak 8.96 11.45 17.61 29.23 46.22 Peak SD 0 0.8 1.64 2.034.13 tt Peak 33.72 29.04 25.04 21.2 18.19 tt Peak SD 0 0.67 0.67 0.170.5 Start Tail 75 75 69 57 43.5 Start Tail SD 0 0 2 10 0.5 Edited 1 0 01 0

TABLE 79 (Run #2) MOD-5014 Derivative Values for FIG. 61 Lag time 30.0520.2 15.36 12.19 10.18 Lag time SD 0 0.17 0.33 0.5 0.17 ETP 118 179300.5 406.5 602.5 ETPSD 0 1 18.5 19.5 111.5 Peak 4.36 7.2 12.86 19.828.55 Peak SD 0 0.18 0.42 0.73 3.15 tt Peak 46.74 35.55 28.88 24.5421.37 tt Peak SD 0 0.83 0.17 0.17 0.33 Start Tail 76 76 74.5 67.5 69.5Start Tail SD 0 0 0.5 0.5 5.5 Edited 1 1 0 0 0

A dose-dependent response was observed following the addition of the twocompounds. At low concentrations of 1.25-2.5 μg/ml, which presumablymimic 40 and 80 μg/kg, respectively, a poor TG was observed, asreflected by increased lag and reduced ETP (FIGS. 56, 57, 60 and 61).Although the higher concentration provided a pronounce improvement inthe TG profile, none of the tested compounds were able to provide acomplete restoration of the TG as obtained with FVIII (FIGS. 71 and 72;Table 80).

TABLE 80 (Derivative values for FIG. 72) FVIII + Low TF FVIII Lag time6.17 11.33 Lag time SD 0.17 0.33 ETP 1869 1604 ETP SD 1 48 Peak 323.34233 Peak SD 13.43 17.69 tt Peak 8.67 15.5 tt Peak SD 0 0.17 StartTail27.5 33.5 Start Tail SD 0.5 0.5 Edited 0 0

These results are in line with published studies and suggest that hFVIIais less effective as a bypassing agent than other replacement therapiesfor thrombin correction, demonstrated by lower TG peak, ETP, and otherparameters. Overlay analysis (FIGS. 58 (A-E), 59 (A-D), 62 (A-E) and 63(A-D)) suggested a slight reduction in MOD-5014 TG response as comparedto NovoSeven®, reflected mainly as increased lag time, and a lowerthrombin peak (estimated as 30-40% lower than in NovoSeven®). This mightbe a consequence of reduced molar content of FVIIa in MOD-5014 whenspiking on mass base rather than on molar base as MOD-5014 consists of83.4% FVIIa with 3 CTP cassettes attached at the C terminus. The twoindependent runs were consistent with each other, providing similarresults with minor variation.

Comparative Thrombin Generation at low TF concentrations

When severe hemophilia A pooled plasma was spiked with low levels of TF,a background TG response was observed that increased as MOD-5014 andNovoSeven® concentrations increased in the tested samples. (FIGS. 64,65, 66(A-E), 67, 68, 69(A-E). 70(A-C): Tables 81-84)

TABLE 81 (NovoSeven ® Derivative values for FIG. 64) 1.25 2.5 5 10 15Control μg/ml μg/ml μg/ml μg/ml μg/ml Lag time 7.33 4.33 4.17 4.17 4.174.17 Lag time SD 0 0 0.17 0.17 0.17 0.17 ETP 592 880 903 1003.5 1256.51350 ETP SD 0 83 34 63.5 77.5 72 Peak 28.6 46.23 51.30 62.16 77.12 92.79Peak SD 0.81 0.74 0.87 3.23 1.02 5.15 tt Peak 17.33 12.83 12.33 12.33 1211.5 tt Peak SD 0.67 0.17 0 0 0 0.17 StartTail 59 64 57 44.5 49 64 StartTail SD 1 0 7 0.5 2 0 Edited 0 1 1 0 0 1

TABLE 82 (MOD-5014 Derivative values for FIG. 65) 1.25 μg/ml 2.5 μg/ml 5μg/ml 10 μg/ml 15 μg/ml Lag time 4.33 4.17 4.17 4.33 4.17 Lag time SD 00.17 0.17 0 0.17 ETP 699.5 822.5 912 1088 1223.5 ETP SD 6.5 39.5 11 3126.5 Peak 40.28 49.32 53.53 64.13 72.19 Peak SD 0.41 1.91 1.72 3.65 2.76tt Peak 12.67 12.33 12.83 12.83 12.17 tt Peak SD 0.33 0 0.17 0.17 0.12StartTail 50.5 60.5 47.5 48 49.5 Start Tail SD 1.5 3.5 2.5 1 1.5 Edited0 1 0 0 0

TABLE 83 (NovoSeven ® Derivative values for FIG. 67) 1.25 2.5 5 10 15Control μg/ml μg/ml μg/ml μg/ml μg/ml Lag time 6.17 3.5 3.67 3.67 3.673.67 Lag time SD 0.17 0.17 0 0 0 0 ETP 526.5 671.5 790.5 795 928 955 ETPSD 7.5 13.5 11.5 5 27 44 Peak 25.34 40.33 46.66 53.84 61.35 65.69 PeakSD 1.4 0.4 0.47 0.13 0.18 0.66 tt Peak 16 11.83 11.5 11.5 11.67 11.33 ttPeak SD 0.33 0.12 0.17 0.17 0 0 Start Tail 56.5 53.5 56 36.5 41 44 StartTail SD 1.5 0.5 8 2.5 2 2 Edited 0 0 1 0 0 0

TABLE 84 (MOD-5014 Derivative values for FIG. 68) 1.25 μg/ml 2.5 μg/ml 5μg/ml 10 μg/ml 15 μg/ml Lag time 4 1.83 4 4 3.83 Lag time SD 0 0.17 0.330.33 0.5 ETP 587 657.5 723.5 761 833.5 ETP SD 11 21.5 49.5 4 94.5 Peak35.35 38.62 42.52 49.28 51.34 Peak SD 0.15 1.51 4.87 3.88 7.12 tt Peak11.83 12.17 13 12.67 12.33 tt Peak SD 0.17 0.17 0.67 1 1 Start Tail 4852 51 39.5 64 Start Tail SD 1 2 6 7.5 0 Edited 0 0 0 0 1

A similar pattern of dose-dependent response was observed in this studyfor both products; however, larger amplitude was obtained due toresponse enhancement by TF. Again, MOD-5014 demonstrated reducedactivity that was more pronounced in comparison to the re-calcificationsstudy, requiring a higher MOD-5014 concentration to provide a suitableoverlay between the two (FIG. 66 (A-E) and FIG. 70 (A-C)). To study thisfurther, an in vitro study was performed further investigating MOD-5014and FVIIa affinity to TF and its effect on TG, as described below.

Conclusions:

Both products demonstrated a dose-dependent TG response when spiked tosevere hemophilia A pooled plasma, with an initial poor response atconcentrations mimicking clinical dose of 40-80 μg/kg. These resultspresumably suggest that doses lower than 40-80 μg/kg will not provide anadequate in vivo response. Furthermore, MOD-5014 demonstrated a reducedTG performance when spiked at a similar concentration as NovoSeven®,suggesting that a slightly increased concentration (30-40%) might beneeded in the clinical setting to provide proper initial hemostaticeffect which is comparable to that of NovoSeven®.

Objective (II): Comparative In Vitro Assessment of MOD-5014 andNovoSeven® Affinity to TF

FVIIa seems to have at least two independent effector mechanisms: thetissue factor (TF)-dependent FVIIa-mediated activation of factor X (FX),which is the classic inducer of the extrinsic pathway of coagulation,and a TF-independent activity of high-dose FVIIa on endogenousphospholipid (PL) surfaces of monocytes or platelets. MOD-5014demonstrated reduced activity when compared to NovoSeven® that was morepronounced in the presence present of TF. Higher MOD-5014 concentrationswere required to provide a suitable overlay between the two compounds(FIG. 66 (A-E) and FIG. 70 (A-C)). Although it was previously reportedthat MOD-5014 affinity to TF was similar to NovoSeven® as measured bySPR and in vitro activation assays, the objective of this study was tofurther investigate this in vitro affinity of MOD-5014 and FVIIa to TFby TG.

MOD-5014 and NovoSeven® were spiked at an escalating range ofconcentrations to severe hemophilia A pooled plasma, in the presence ofescalating concentrations of TF. The study was repeated twice, and theresults of each run are provided in FIGS. 73-88 and Tables 85-90.

TABLE 85 (Derivative Values for FIG. 73) NovoSeven MOD-5014 TF TF TF TFTF TF TF TF TF TF 0 pM 0.5 pM 1 pM 2.5 pM 5 pM 0 pM 0.5 pM 1 pM 2.5 pM 5pM Lag time 35.67 3.67 2.33 2.53 1.33 36 4.67 2.67 2.33 1.33 Lag time SD0 0 9 6 0 0 0 0 0 0 ETP 0 326 460 1038 1654 0 264 484 1117 1598 ETP SD1- 0 0 0 0 1- 0 0 0 0 Peak 10.73 12.88 22.47 95.78 233.67 8.23 9.04 27.499.11 231 Peak SD 0 0 0 0 0 0 0 0 0 0 tt Peak 51.67 19.33 12 7.67 5 5622.33 10.33 8 4.67 tt Peak SD 0 0 0 0 0 0 0 0 0 0 StartTail 0 75 58 3325 0 75 49 33 25 Start Tail SD 1- 0 0 0 0 0 0 0 0 0 Edited 0 0 0 0 0 0 00 0 0

TABLE 86 (Derivative Values for FIG. 75) NovoSeven MOD-5014 TF TF TF TFTF TF TF TF TF TF 0 pM 0.5 pM 1 pM 2.5 pM 5 pM 0 pM 0.5 pM 1 pM 2.5 pM 5pM Lag time 25.67 5.33 3 2.67 1.67 50 6.67 3 2.67 1.67 Lag time SD 0 0 00 0 0 0 0 0 0 ETP 0 338 503 1054 1092 0 245 448 1034 1554 ETP SD 1- 0 00 0 1- 0 0 0 0 Peak 11.8 14.13 26.41 98.73 252.42 9.39 10.27 22.59 93.16240.75 Peak SD 0 0 0 0 0 0 0 0 0 0 tt Peak 41 19.67 12.33 8 4.67 56.3522 12 8.35 5 tt Peak SD 0 0 0 0 0 0 0 0 0 0 StartTail 0 72 55 24 24 0 6456 34 23 Start Tail SD 1- 0 0 0 0 1- 0 0 0 0 Edited 0 0 0 0 0 0 1 0 0 0

TABLE 87 (Derivative values for FIG. 77) NovoSeven MOD-5014 TF TF TF TFTF TF TF TF TF TF 0 pM 0.5 pM 1 pM 2.5 pM 5 pM 0 pM 0.5 pM 1 pM 2.5 pM 5pM Lag time 18 8 3.33 2.67 1.67 27.67 5.67 5 3 1.67 Lag time SD 0 0 0 00 0 0 0 0 0 ETP 0 418 858 1111 1527 235 344 490 982 1514 ETP SD 0 0 0 00 0 0 0 0 0 Peak 16.85 18.67 36.73 117.89 254.79 19.41 13.22 28.28 94.2261.12 Peak SD 0 0 0 0 0 0 0 0 0 0 tt Peak 31 20 12 7.53 4.67 40.6721.53 10.67 8.33 4.67 tt Peak SD 0 0 0 0 0 0 0 0 0 0 StartTail 75 69 5929 22 26 75 50 38 21 Start Tail SD 0 0 0 0 0 0 0 0 0 0 Edited 0 0 0 0 01 0 0 0 0

TABLE 88 (Derivative values for FIG. 81) NovoSeven MOD-5014 TF TF TF TFTF TF TF TF TF TF 0 pM 0.5 pM 1 pM 2.5 pM 5 pM 0 pM 0.5 pM 1 pM 2.5 pM 5pM Lag time 17 7.33 3.67 3 1.67 20.33 8.67 4 3 1.67 Lag time SD 0 0 0 00 0 0 0 0 0 ETP 717 718 665 1012 1633 197 460 754 1059 1602 ETP SD 0 0 00 0 0 0 0 0 0 Peak 23.87 28.73 41.13 90.03 236.54 16.38 19.99 32.2 92.24236.43 Peak SD 0 0 0 0 0 0 0 0 0 0 tt Peak 29.33 19 13.67 8.33 5 32.6721 13.67 8.67 5 tt Peak SD 0 0 0 0 0 0 0 0 0 0 StartTail 76 64 45 34 2376 72 52 34 25 Start Tail SD 0 0 0 0 0 0 0 0 0 0 Edited 1 1 0 0 0 0 0 10 0

TABLE 89 (Derivative values for FIG. 82) NovoSeven MOD-5014 TF TF TF TFTF TF TF TF TF TF 0 pM 0.5 pM 1 pM 2.5 pM 5 pM 0 pM 0.5 pM 1 pM 2.5 pM 5pM Lag time 25 7.33 3.33 2.67 1.67 32.67 6 3.33 2.67 1.67 Lag time SD 00 0 0 0 0 0 0 0 0 ETP 0 333 734 1230 1670 0 117 760 1107 1605 ETP SD 1-0 0 0 0 1- 0 0 0 0 Peak 13.5 13.45 26.75 136.91 252.5 10.71 13.15 30.41105.24 244.68 Peak SD 0 0 0 0 0 0 0 0 0 0 tt Peak 40.67 23.33 14.33 7.674.67 50 21 13 8.35 4.67 tt Peak SD 0 0 0 0 0 0 0 0 0 0 StartTail 0 72 7533 24 0 75 61 31 24 Start Tail SD 1- 0 0 0 0 1- 0 0 0 0 Edited 0 0 0 0 00 0 1 0 0

TABLE 90 (Derivative Values of FIG. 83) NovoSeven MOD-5014 TF TF TF TFTF TF TF TF TF TF 0 pM 0.5 pM 1 pM 2.5 pM 5 pM 0 pM 0.5 pM 1 pM 2.5 pM 5pM Lag time 21.33 7 4 2.67 2 26.33 7 3.67 2.67 2 Lag time SD 0 0 0 0 0 00 0 0 0 ETP 388 459 679 1105 1581 302 354 754 1607 1531 ETP SD 0 0 0 0 00 0 0 0 0 Peak 16.18 21.97 30.22 109.4 260.86 12.28 14.34 28.58 103.67254.16 Peak SD 0 0 0 0 0 0 0 0 0 0 tt Peak 34.33 19 14.33 7.67 4.6741.67 21.33 12.53 7.67 4.67 tt Peak SD 0 0 0 0 0 0 0 0 0 0 StartTail 7364 58 32 23 74 52 69 30 25 Start Tail SD 0 0 0 0 0 0 0 0 0 0 Edited 1 11 0 0 1 0 1 0 0

Spiking with increasing concentrations of TF with fixed doses of eitherNovoSeven® or MOD-5014 provided a TF-dependent increase in TGperformance, as reflected by reduced lag time and increased ETP andthrombin pick (FIGS. 79 (A-C), 80 (A-C), 87 (A-E) and 88 (A-E)). Forboth compounds at all doses and TF concentrations below 5 pM, poor tomoderate TG response was observed as reflected by increased lag time andreduced ETP. A higher concentration of TF (5 pM) provided a pronouncedimprovement in the TG profile (FIGS. 79 (A-C), 80 (A-C), 87 (A-E) and 88(A-E)). However, none of the tested compounds were able to provide acomplete restoration of the TG as obtained with FVIII (FIGS. 71 and 72,and Table 82). Interestingly, increasing the concentrations ofNovoSeven® or MOD-5014 at constant TF levels did not improve TGperformance (FIGS. 79 (A-C), 80 (A-C), 87 (A-E) and 88 (A-E)), furtheremphasizing the importance of TF for the proper activity of bothcompounds.

Comparison of the TG profiles of MOD-5014 and NovoSeven® by overlayanalysis (FIGS. 74 (A-E), 76 (A-E), 78 (A-E), 82, 85 (A-C), 86 (A-E);Table 89) suggested a slight reduction in MOD-5014 response (estimatedas 20-30% lower) without TF or at very low TF concentration (0.5 pM). Asthe level of TF increases, a similar response is observed at allMOD-5014 and NovoSeven® concentrations on both repeats (FIGS. 74 (A-E),76 (A-E), 78 (A-E), 82, 85 (A-C), 86 (A-E); Table 89).

Conclusion:

This study further confirms the results in vitro by spiking bothcompounds at fixed concentrations in the presence of escalatingconcentrations of TF. The amount of TF in the sample was predominantlyresponsible for the increased TG response, further confirming abiological similarity between MOD-5014 and NovoSeven®.

Objective (III) Comparative Assessment of MOD-5014 and NovoSeven®Activity Using Rotation Thromboelastography (ROTEM)

Over the last decade, thromboelastography emerged as a valuable tool formonitoring hemostasis in coagulopathy, blood transfusion and clottingfactor replacement therapy. In this respect, thromboelastography hasbeen used in hemophilia, factor VIII or IX replacement therapy, andassessment of the effect of aFVIIa in hemophilia patients withinhibitors. ROTEM may be used for assessing of the effects ofcoagulation and anti-fibrinolytic agents in thrombocytopenia,Glanzmann's thrombasthenia and hemodilution. The objectives of the studywere to compare the in vitro ROTEM performance of MOD-5014 andNovoSeven® in severe hemophilia A plasma at a range of concentrationswhich correlate to the proposed clinical doses, and assess the minimaleffective dose as part of the preparations for the FIH study.

The results of the experiments are presented in the Tables 91 and 92.

TABLE 91 Spiking FVIII-Deficient Plasma with Factor VIII Clot timeα-angle MCF Test Treatment (CT) (sec) (degrees) (mm) NATEM Intact Noclot No clot No clot FVIII 1359 17 15 INTEM Intact 1123 9 11 FVIII 33572 14

Table 91 shows plasma clotting without FVIII and following plasmaspiking with FVIII (1 U/mL), which was used as control compound in thestudy. In the NATEM test, no clotting was observed in the intact plasma,while spiking the plasma samples with FVIII was followed by slight clotformation. In contrast, in the INTEM test clot formation occurred inboth intact and treated plasma, whereas in the latter it was muchstronger (CT was shorter and the α-angle was 8-fold higher). Maximumclot firmness (MCF) was slightly increased. These results suggest thatthe intrinsic system (INTEM) may be a useful test for the assessment ofFVIII-replacement in hemophilia A.

TABLE 92 The Effect of MOD-5014 and Novo Seven on Clot Formation inFVIII-Deficient Plasma Conc. Clot time α-angle MCF Test Treatment(μg/mL) (sec) (degrees) (mm) NATEM Control — No clot No clot No clotNovoSeven 2.5 No clot No clot No clot 10 nd nd nd 15 1100 20 16 MOD-50142.5 No clot No clot No clot 10 1601 10 12 15 1244 14 15 INTEM Control —1123 9 11 NovoSeven 2.5 810 12.5 10 10 531 25 13 15 472 26 12 MOD-50142.5 797 14.5 12 10 611 22.5 14.5 15 660 27 13 EXTEM Control — 404 19.510.5 NovoSeven 2.5 371 20.5 12.5 10 340 27 13 15 377 30 14 MOD-5014 2.5355 22.5 12.5 10 324 28.5 14 15 323 30 13.5 nd—not determined

Table 92 shows the effects of FVIIa on clot formation in FVIII-deficientplasma.

Recalcification with Contact Activation of Plasma (INTEM Test):

CT was shorter and the α-angle increased gradually in plasma treatedwith both types of FVIIa at 2.5 and 10 μg/mL compared to non-treatedplasma. By increasing FVIIa concentration to 15 μg/mL, the changes inclot formation did not differ from that of plasma treated with 10 μg/mLFVIIa. No difference was found between the activities of MOD-5014 andNovoSeven.

Recalcification with Activation of the Extrinsic Pathway (EXTEM Test):

There was a decrease in CT and increase of MCF in both MOD-5014 andNovoSeven-treated plasma compared to non-treated plasma. An increase inclot propagation (α-angle) was observed to a similar extend in plasmatreated with both agents at 2.5 and 10 μg/mL, with no further change at15 μg/mL.

Conclusions:

INTEM appeared to be the most reliable test among the different ROTEMtests utilized to compare the activity of MOD-5014 and NovoSeven. A doseof 2.5 μg/ml, which mimics an in vivo dose of 80 μg/kg, resulted in alow response for both compounds as reflected by slightly decreasedclotting time, and increased α-angle compared to non-treated plasma,tested utilizing both INTEM and EXTEM. Increasing concentrations ofMOD-5014 and NovoSeven® to 10 and 25 μg/ml was followed bydose-dependent stimulatory effect on clot formation. The results alsodemonstrate no essential difference in the effects of either agent onclot formation, providing very similar ROTEM values at both agents.

Example 14 Assessment of MOD-5014 Pharmacokinetics, Pharmacodynamics andCorrection of Hemophilic Coagulopathy in Dogs with Hemophilia a (FVIIIDeficiency)

Study Objective—

The objective of the current study was to evaluate and characterizeMOD-5014 pharmacokinetics, pharmacodynamics, and correction ofhemophilic coagulopathy in dogs with severe hemophilia A (FVIIIdeficiency, <0.01% FVIII).

Justification of Test System

The Chapel Hill hemophilia A dog colony has the advantage of nodetectable FVIII activity in FVIII bioassays and chromogenic assays, andlittle or no FVIII antigen by ELISA (<0.005 U/ml). This model has beenused in the past as part of the non-clinical pharmacologicalcharacterization of different clotting factors. It has been proposedthat this model can provide an accurate recapitulation of human biologywithin the particular area of hemostasis, and provide comparable PK/PDparameters to those previously observed in human studies. In addition,TEG, which is used as a screening tool assessing overall hemostaticdysfunction or correction in clinical setting has been shown to be apredictive tool in this model as well suggesting further correlationbetween human and dog hemostasis.

Materials and Methods

Materials—

MOD-5014: 2.6 mg/ml, stored frozen (−60 to −80° C.). No dose formulationpreparation was required. Materials were thawed to room temperatureprior to administration.

The pre-formulated dosing formulation was handled aseptically andadministered as received; no dose formulation preparation was required.Test article (MOD-5014) was thawed only once prior to dosing. The testarticle was removed from frozen storage and thawed to room temperatureprior to administration.

Method: Ex vivo phase:

The ex vivo phase was conducted according to Knudsen et al 2011,Haemophilia. 17(6):962-970, in order to establish the minimal effectivedose which would support dose selection for the in vivo study.

During the first phase, dose dependent ex vivo Whole Blood Clotting Time(WBCT) and Thromboelastography (TEG) assessments of MOD-5014 spiked intofresh blood, from two individual FVIII deficient dogs, were performed.The lowest dose of MOD-5014, which significantly improved the TEG andWBCT performance, was considered the minimal effective dose. Based onthe first phase and the establishment of the minimal effective dose, thedoses for the in vivo second phase were selected.

Fresh canine blood was drawn from two individual FVIII hemophilic dogsand analyzed independently in order to confirm reproducibility ofresults. Briefly, 15 mL of canine blood were drawn into a syringe,transferred to a conical tube and spiked with MOD-5014 to finalconcentrations of 0.568, 1.136, 2.273, 4.545, and 9.909 μg/mL, whichcorrelates to anticipated in vivo Cmax following administration of 50,100, 200, 400, 800 μg/kg of MOD-5014 in FVIII deficient dogs assumingthe following: Canine estimated weight is 20 kg and Blood volume foreach animal is 40 mL/lb (88 mL/kg).

Blood from these spiking was assayed within <5 min of adding the testmaterial as follows:

Step 1: Whole Blood Clotting Time (WBCT): 1 mL of blood spiked withMOD-5014 to designated final concentrations was used for the WBCT.Briefly, the whole blood clotting time (WBCT) is a modification of theLee-White clotting time using two siliconized glass tubes (Vacutainer™#6431, Becton-Dickinson, Rutherford, N.J.) in a 28° C. water bath. OnemL of whole blood spiked with MOD-5014 at designated concentrations wassplit equally between two siliconized tubes. A timer was started. Afterone minute, one tube was tilted every 30 sec, the other leftundisturbed. When a clot was formed in the tilted tube, the second tubewas then tilted every 30 sec until a clot formed. The time for formationof a fully gelled clot in the second tube was recorded as the WBCT. Innaïve hemophilia A dogs, the WBCT is generally >40 minutes but canbe >60 min. The test is stopped if the value exceeds 60 min.

Step 2: Thromboelastography: 1 mL of blood spiked with MOD-5014 todesignated final concentrations was mixed with Kaolin (Lot # is providedby Haemoscope), 360 μl of this mixture was placed in the cup fortesting. TEG recordings were allowed to proceed for approximately 90min. A typical range of TEG values is provided in the Table 93 below.

TABLE 93 Thromboelastography Typical Range of Values TEG R K Angle MA(Kaolin activated) (min) (min) (degrees) (mm) Normal (n = 2) 7.4 ± 1.11.6 ± 0.2 67.2 ± 2.9 61.4 ± 20.6 Hemophilia A (n = 2) >60 NA NA NA

The lowest dose that demonstrates significant clotting improvement willbe used as the initial dose for the in vivo study, proposing propermonitoring of blood clotting and hemostasis as measured during the phaseII of the study and by utilizing the different assays as describedbelow.

Method: In Vivo Phase:

Six naïve mixed breed Chapel Hill hemophilia A dogs (Canis familiaris)weighing approximately 20 kg were used in this study. Dogs received IVdoses of 50 μg/kg (N=2), 200 μg/kg (N=4), 400 μg/kg (N=2), or 600 μg/kg(N=2) MOD-5014. Doses were given as a 5 minute IV infusion. Bloodsamples were collected for concentration and activity measures bytranscutaneous puncture of the cephalic vein prior to dosing, and at0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 32, 48, 72 and 96 hours post-dosing.Four dogs were administered twice with MOD-5014 during a period of up to8 days, and 2 additional dogs were administered once. The study wasdesigned as a dose-staggered escalating study, i.e. following eachinjection in-time clotting analysis determined the timing of the nextdose.

During the in vivo phase, MOD-5014 pharmacokinetics and pharmacodynamicswere analyzed, and WBCT, thromboelastography, aPTT, basic clinicalpathology, and a clinical observation (based on animal behavior) wereassessed. Bioanalytical assessment was conducted for the test article.

In vivo Phase—Part A

During the first part, 2 naïve dogs were injected on day 1 with aninitial dose of 50 μg/kg MOD-5014 and monitored for WBCT and TEGperformance. Once these values returned to baseline (pre-dose), a seconddose of 200 μg/kg MOD-5014 was injected. Table 94 presents the outlineof Part A.

TABLE 94 Study design Part A injection injection Animal Dose Ani- TestInjection Dose Dose Weight Volume mal Article time point Route (μg/kg)(Kg) (mL/animal) N06 MOD- T = 0 IV 50 20 0.384 5014 N06 MOD- T = 48 hrIV 200 20 1.538 5014 (post ini- tial dose) P14 MOD- T = 0 IV 50 22.60.434 5014 P14 MOD- T = TBD IV 200 22.6 1.736 5014 (post ini- tial dose)

In vivo Phase—Part B

During the second part and following the completion of the Part A, twoadditional naïve dogs were injected on day 1. The animals were againmonitored for WBCT and TEG performance. Once those values returned tobaseline (pre-dose), a second dose was injected. Table 95 presents theoutline of Part B.

TABLE 95 Study design Part B injection injection Animal Dose Ani- TestInjection Dose Dose Weight Volume mal Article time point Route (μg/kg)(Kg) (mL/animal) Blondie MOD- T = 0 IV 200 20.8 1.6 5014 Blondie MOD- T= 48 IV 400 20.8 3.2 5014 (post ini- tial dose) Josie MOD- T = 0 IV 20020.5 1.576 5014 Josie MOD- T = 48 IV 400 20.5 3.15 5014 (post ini- tialdose)

In vivo Phase—Part C

During Part B and following the completing of Part A, two additionalnaïve dogs were injected on day 1 (Table 96).

TABLE 96 Study design of Part C injection injection Animal Dose Ani-Test Injection Dose Dose Weight Volume mal Article time point Route(μg/kg) (Kg) (mL/animal Joanie MOD- T = 0 IV 600 20.6 4.75 5014 N05 MOD-T = 0 IV 600 18.4 4.24 5014

Justification for Route of Administration

The intravenous route is the intended route of administration of thistest article in humans.

Justification of Dose Levels

The dose levels were selected on the basis of the proposed dose rangewhich was previously tested for rhFVIIa and the dose range consideredfor further assessment in the Phase I study.

In order to assess the hemostatic potential of MOD-5014 which willenable the prediction of the minimal effective dose in Hemophilia A dogsprior to infusion, a range of doses was evaluated in WBCT and TEGspiking studies at a dose range that approximates the maximal expectedlevel following in vivo IV administration. In both assays, spiking ofMOD-5014 at the lowest dose (0.568 μg/mL, which corresponds to 50 μg/kg)demonstrated a minor improvement in hemostatic effect that improved asconcentrations increased but without a complete normalization of TEGvalues, in line with Knudsen et al., 2011. Based on the obtained resultsthe lowest dose to be further studied in vivo was the lowest assesseddose i.e. 50 μg/kg.

Administration

Administration was by IV. The test article was administered up to 2times during 8 days. Approximately 10% of the test article was injectedover ˜1 min and the dog was observed for any obvious clinical reaction(e.g., urticaria and listlessness). When the dog was considered astolerant to the injection, the remaining 90% was infused over 1 to 5minutes. The injection time and volume were recorded.

Different doses were provided by varying the dose volume. Administrationof test article was followed by a saline flush.

The animals were fasted prior to dosing.

Study Evaluations

Physical Examinations:

dogs underwent a general examination prior to study entry, and onlythose with normal general examinations were included.

Cage side observations: any concerns regarding the animal's behavior orgeneral health noted in the clinical examination of the animal wererecorded.

Detailed clinical observations: During the infusion phase of the study,blood sampling was performed.

Body weights were recorded at the start of the study and between Days 7and 14 following study initiation.

Food consumption: Dogs were fed their usual amount of food on a dailybasis and observed for food consumption.

Clinical Pathology

Clinical pathology evaluations were conducted on study animals atpre-dose. Platelet counts, WBC, HCT, and HGB were performed by FOBRL.Samples were collected for further clinical chemistry tests should anyclinical events have occurred.

Plasma Analysis

Sample Collection and Handling

All blood samples were taken by transcutaneous puncture of the cephalicvein using a 21G or comparable butterfly needle. 10 mL of blood wascollected in a 10 mL syringe containing 1 mL anticoagulant (3.2% sodiumcitrate [0.12M]). The sodium citrate was diluted 1:10 as the blood wascollected, according to standard lab protocol. The 10 mL blood samplewas transferred from the syringe to a 15 mL polypropylene conical tubeand gently inverted to ensure mixing without hemolysis. The blood wasthen centrifuged at 3000 g for fifteen minutes without brake at 4° C.Following centrifugation, the plasma supernatant was transferred intonew 15 mL polypropylene conical tube and centrifuged at 3000 g withoutbrake at 4° C. for an additional 7 minutes, to ensure sufficientseparation of plasma from other blood materials. After the secondcentrifugation, plasma from each sample was transferred by apolypropylene pipette to a polypropylene tube and immediately placed ina −80° C. freezer.

Multiple aliquots of 100 μL of plasma were transferred to Micronicstubes (or comparable) and frozen rapidly (−80° C.). Except for WBCT andTEG, all assays (enzyme-linked immunosorbant assay [ELISA], activatedpartial thromboplastin time [aPTT], and thrombin generation assay [TGA])were performed in batch for all animals after the infusion and samplingwere completed.

MOD-5014 ELISA

MOD-5014 ELISA was performed utilizing an assay that specifically andselectively detects MOD-5014, utilizing a commercially availableanti-FVIIa antibody (Ab) and an in-house polyclonal anti-CTP Ab.Citrated plasma samples were collected at pre-dosing (i.e. baseline), 15min, 30 min, 1, 2, 4, 6, 8, 12, 24, 32, 48, 72, and 96 hours.

FVIIa Clotting Activity

FVIIa clotting activity was measured utilizing the commerciallyavailable STACLOT VIIa-rTF kit (Ref #00281, Stago) adjusted toquantitate FVIIa activity in hemophilic dog matrix.

WBCT

The WBCT assays were performed by a two-tube procedure at 28° C., andtested after collection. One mL of whole blood was collected with 1 mLsyringe and was divided equally between two siliconized tubes(Vacutainer™, #6431, Becton-Dickinson, Rutherford, N.J.). The first tubewas tilted every 30 sec. Following clot formation, the second tube wastilted and observed every 30 sec. The endpoint was the clotting time ofthe second tube. In this study, WBCT was performed by FOBRL atpre-dosing (i.e. baseline), 15 min, 1, 2, 4, 6, 8, 12, 24, 48, 72, and96 hours, or until baseline values were reached.

TEG

Blood for TEG was drawn at each sampling point and tested at FOBRL/UNCwithin 2 min post-collection using the Haemoscope TEG 5000Thromboelastography Analyzer according to the manufacturer'sinstructions. Briefly, the first 3 mL of blood were discarded, then 1 mLof blood was drawn and mixed with Kaolin (Lot #A-30-05, provide byHaemoscope). 360 μL of this premixed blood/initiator was placed in theinstrument and analyzed. TEG recordings were allowed to proceed forapproximately 60-90 min. The test was performed at pre-dosing (i.e.baseline), 15 min, 1, 2, 4, 6, 8, 12, 24, 48, 72, and 96 hours, or untilbaseline values were reached.

aPTT

aPTT was determined at FOBRL/UNC using the ST4 coagulation instrument(Diagnostica Stago, Asnieres, France). The test mixture consisted ofequal portions of partial thromboplastin reagent (Triniclot, DiagnosticaStago), 0.025 M CaCl₂ and citrated test plasma. Samples were tested atpre-dosing (i.e. baseline), 15 min, 1, 2, 4, 6, 8, 12, 24, 48, 72, and96 hours.

Data Analysis

Non-compartmental analysis and pharmacokinetic modeling were performedon individual animal data with Phoenix WinNonlin version 6.3 (PharsightCorporation, Sunnyvale, Calif.). Analysis was performed on MOD-5014plasma concentration data as well as on activity data assuming aninitial specific activity of 15,563 units/mg.

For non-compartmental analysis a program for IV infusion was used. Thearea under the curve from time zero to the last measureable time point(AUC_(0-t)) was estimated using the trapezoidal method. Log-linearregression over the last three or more time points was used to estimatethe elimination constant (λ) which was used to estimate the terminalhalf-life (t_(1/2)) and AUC from zero to infinity (AUC_(0-∞)) from thefollowing equations:

t _(1/2)=ln(2)λ

AUC _(0-∞) =AUC _(0-t) +C _(t)/λ

where Ct is the last measureable concentration. Plasma clearance (CL)was calculated from dose divided by AUC_(0-∞). The maximum concentration(Cmax) and the time it was observed (Tmax) were determined directly fromthe data. Since the analysis was based on an IV infusion, theconcentration at the start of infusion was set to 0 and an initialvolume of distribution was not calculated.

For pharmacokinetic modeling, various models and fitting strategies wereinvestigated including evaluation of one-, two-, and three-compartmentmodels and different weighting schemes.

Models were evaluated based on the correlation of observed and predictedconcentrations, the relative values of parameter estimates and errorestimates, and the Akaike Inclusion Criterion, a mathematical evaluationfor comparing one model to another.

Both plasma concentration and activity versus time data were describedbest by a twocompartment model. Concentration data were weighted by theinverse of the square of the predicted concentration and activity datawere weighted by the inverse of the predicted concentration. Theweighting schemes prevent the early, high concentrations from exertingtoo much influence over the curve fit. The model is illustrated in FIG.90.

The model generates estimates for the volume of distribution of thecentral compartment (V1), the elimination rate constant k10 and theinter-compartmental rate constants, k12 and k21. The central compartmentis the compartment into which drug is administered and from whichsamples are collected, in this case, vascular space. The rate constantsassociated with the distribution and elimination phases of the curve, αand β, are calculated from to intercompartmental rate constants. Otherparameters calculated from the primary parameters include AUC, Cmax,T_(max), CL, MRT and the half-lives associated with the distribution andelimination phases of the curve (t_(1/2α), t_(1/2β)).

Results Ex vivo Study Results

WBCT

WBCT was measured following spiking of MOD-5014 to the blood of twoindividual hemophilic A dogs, at final concentrations ranging from0-9.09 μg/mL, which are calculated to be equivalent to in vivo Cmaxfollowing administration of 50, 100, 200, 400, 800 μg/kg of MOD-5014,respectively. The baseline (pre) WBCT value was different between thetwo dogs, but within the acceptable range for Hemophilia A animals (seeTable 97).

TABLE 97 WBCT Following MOD-5014 Spiking Canine Blood Conc. WBCT Conc.WBCT Dog ID (μg/mL) (min) Dog ID (μg/mL) (min) P22 0 (Pre) 41.5 O93 0(Pre) 52 P22 0.568 37 O93 0.568 42.5 P22 1.136 34.5 O93 1.136 45.5 P222.273 33.5 O93 2.273 41.5 P22 4.545 35.5 O93 4.545 40 P22 9.09 32.5 O939.09 34

Significant reduction in WBCT was observed as MOD-5014 concentrationincreased with optimal correction (˜30 min, based on internal laboratoryassessment) at the highest range of MOD-5014 concentrations, aspresented in Table 97 and FIG. 89.

For Dog P22, a lower WBCT baseline was observed and lower MOD-5014concentrations were effective, while Dog 093 required higher MOD-5014concentrations to reach the target WBCT value due to its higher baselinevalue.

Thromboelastography

MOD-5014 at a range of doses was spiked independently into whole bloodfrom two Hemophilia A dogs and was used to simulate a relevantconcentration in blood shortly after IV injection of doses ranging from50-800 μg/kg. Kaolin-TEG parameters were improved at a dose dependentmanner with some fluctuation and variability between the two individualsdue to assay variability or technical error (Table 98).

TABLE 98 Effect of MOD-5014 spiked in canine hemophilia blood assessedby Kaolin-activated TEG Thromboelastography (Kaolin) Dog Conc. R K MADog Conc. R K MA ID (μg/mL) (min) (min) Angle (mm) ID (μg/mL) (min)(min) Angle (mm) P22 0 (Pre) >60.0 — — — O93 0 (Pre) >60.0 — — — P220.568 43.4 23.2 9.3 38 O93 0.568 40.8 13.2 15.1 46.1 P22 1.136 37.2 1217.7 48.4 O93 1.136 38.1 10.1 19.6 36.4 P22 2.273 41.9 24 9.6 49.4 O932.273 32.4 14.5 15.3 40.1 P22 4.545 32.8 12.2 17.7 49.3 O93 4.545 36.414.3 14.8 50.3 P22 9.09 26.8 n/a 12.6 n/a O93 9.09 31.4 9.4 16.7 41.7

The obtained values were comparable to the ones reported at Knudsen etal 2011, for recombinant rhFVIIa, taking to account the reduced specificactivity of MOD-5014 (2.3-2.5 fold reduction in FVIIa specific activity)and lower FVIIa content (72.3%).

Conclusion:

In both assays, the lowest dose of 0.568 μg/mL, which corresponds to 50μg/kg, demonstrated an improvement in hemostatic effect which improvedas concentrations increased without a complete normalization of TEGvalues, aligned with Knudsen et al 2011.

In vivo Study Results:

In-life Clinical Examinations

All infusions were well-tolerated. No injection site reactions werenoted. No significant changes in hemoglobin, hematocrit, white bloodcell count, or platelet counts was noted. No changes in appetite orother behaviors were noted during the study period. Since no clinicalevents occurred during the study period, additional clinical chemistryparameters (e.g. liver enzymes and renal function) were not performed.

Plasma, Pharmacokinetic and Clotting Analysis

The pharmacokinetics data is presented in Tables 99 and 100 below.

FIG. 91 and FIG. 92 present the mean MOD-5014 plasma concentration andactivity over time following IV infusion. Plasma concentrations show aninitial, relatively fast decline over the first 8 to 12 hours followedby a slower decline. Activity declines over time falling below the lowerlimit of the assay (12.6 mU/mL) after about 32 hours in the 50 μg/kggroup and beyond 48 hours in the 200 and 400 μg/kg groups. Activity datafrom the 600 μg/kg dose group at 72 hours and beyond are excluded fromthe analysis because the results were increasing with time and notconsistent between the two animals.

FIGS. 93(A-B) through FIGS. 96(A-B) plot the concentration and activitytogether for each dog.

In the 50 and 200 μg/kg dose groups (FIGS. 93(A-B) and 94(A-D)),concentration and activity followed similar patterns through 8 to 12hours after infusion and then concentrations tended to level off whileactivity continued to decline until it dropped below the level of theassay. In the 400 and 600 μg/kg groups (FIGS. 95(A-B) and 96(A-B)),activity appeared to drop somewhat faster than concentration startingimmediately after infusion. Concentration curves tended to level offwhile activity curves declined faster until they dropped below the assaylimit or, in the case of the 600 μg/kg group, were excluded fromanalysis.

Results of non-compartmental analysis are shown in Table 99 and Table100 for concentration and activity data, respectively. Results ofnon-compartmental analysis indicated that AUC_(0-t) was nearly as largeas AUC_(0-∞) in all cases indicating that the duration of sampling wasadequate to describe the pharmacokinetic and pharmacodynamic profiles.FIGS. 97(A-J) and FIGS. 98(A-J) show the concentration and activityversus time for each dog. The estimated terminal slope is represented onthese graphs by a solid line. Each graph also contains informationrelated to the estimation of the terminal slope including thecoefficient of determination (Rsq and Rsq_adjusted), the number ofpoints used to estimate the slope, and the terminal t_(1/2)(HL_Lambda_z).

With one exception (in the 200 μg/kg group) plasma concentration andactivity levels both were highest at the first time point measured (0.25hr). Both concentration and activity were dose related and approximatelydose proportional as indicated by the Cmax/Dose and CL parametersestimates across dose groups. By both concentration and activitymeasures, CL appeared to be somewhat faster at the lowest dose. At thelowest dose, concentrations and activity dropped below the assay limitsafter 32 hours, possibly limiting the full characterization of thepharmacokinetics at this dose. CL was similar based on concentrationmeasurements compared to activity measurements. The t_(1/2) estimatedbased on concentrations were much longer after the 400 and 600 μg/kgdoses (>40 hours) compared to the 200 μg/kg group (19.3 hours) and the50 μg/kg group (7.76 hours). Terminal t_(1/2) based on activity wasapproximately 3 to 5 hours and similar across dose groups.

Estimates of AUC and CL based on the models agreed very well with thosederived from non-compartmental analysis. The apparent volume ofdistribution derived from the models was approximately 90 to 120 mL/kg(larger in the low dose group) based on plasma concentrations and 40 to70 mL/kg based on activity. The t_(1/2β) estimated by the models forboth plasma and activity measurements were very similar to the terminalt_(1/2) estimated by non-compartmental analysis.

MRT is an estimate of the time an individual drug molecule is incirculation. MRT estimates were consistent across dose groups based onactivity, approximately 4 to 7 hours. Based on concentration, MRT waslonger at the higher doses (approximately 25 hours) compared to the 200and 50 μg/kg groups (16.6 and 11.7 hours, respectively).

Parameters calculated for concentration and activity data bynon-compartmental analysis are summarized in the tables below. With theexception of the 50 μg/kg dose group in which the characterization ofthe pharmacokinetics may have been limited by assay sensitivity, plasmaclearance was similar across dose groups when measured both by plasmaconcentration and by activity.

TABLE 99 Mean MOD-5014 Pharmacokinetic Parameters Based on ConcentrationMeasurements Following IV Infusion in Dogs Estimated byNon-compartmental Analysis Dose C_(max) T_(max) AUC_(0-∞) CL T_(1/2)(μg/kg) (ng/mL) (hr) (hr*ng/mL) (mL/hr/kg) (hr) 50 371 0.250 2230 22.87.76 200 2210 0.313 15000 13.5 19.3 400 4380 0.250 38100 10.7 47.6 6005280 0.250 47000 12.8 45.8

TABLE 100 Mean MOD-5014 Pharmacokinetic Parameters Based on ActivityFollowing IV Infusion in Dogs Estimated by Non-compartmental Analysis CLDose Dose C_(max) T_(max) AUC_(0-∞) (mL/ T_(1/2) (μg/kg) Units/kg(mU/mL) (hr) (hr*mU/mL) hr/kg) (hr) 50 778.15 10800 0.250 29900 26.23.34 200 3,112.6 52100 0.250 172000 18.2 4.72 400 6,225.2 104000 0.250404000 15.6 4.72 600 9,337.8 155000 0.250 700000 13.4 5.22

It was previously reported that, following administration of rhFVIIa tohemophilic dogs, its half-life based on antigen FVIIa level was 3 hr,and based on activity 1.8 hr (Knudsen et al., 2011, summarized below inTable 101).

TABLE 101 Concentration Activity Measurements Measurements CL T_(1/2) CLT_(1/2) (ml/hr/kg) (hr) (ml/hr/kg) (hr) NovoSeven ® 24.5 3 46.1 1.8 (270μg/kg) Knudsen et al. 2011

The attachment of CTP to FVIIa significantly increased the half-life to19.3-45 hr and 4.72-5.22 based on antigen and activity level,respectively, thus extending product half-life by an average of 3-5-foldcompared to reported data. Clearance was affected in a similar manner,and was significantly reduced by 2-3-fold as compared to Knudsen et al.2011.

Whole Blood Clotting Time (WBCT) Analysis

WBCTs were tested after collection by FOBRL at pre-dosing (i.e.baseline), 15 min, 1, 2, 4, 6, 8, 12, 24, 48, with the aim to monitor ituntil baseline values were achieved. As presented in Table 102, theaverage pre-dose WBCT time of all animals was actually at the lowerrange of hemophilic dogs anticipated WBCT (50-35 min).

TABLE 102 WBCT Analysis Time Blondie Blondie Joanie Josie Josie N05 N06N06 P14 P14 (hr) 200 ug/kg 400 ug/kg 600 ug/kg 200 ug/kg 400 ug/kg 600ug/kg 50 ug/kg 200 ug/kg 50 ug/kg 200 ug/kg 0 31 28 30 32.5 39.5 39.5 3730.5 45 40.5 0.25 24.5 24 27 30 31 29.5 31 30 35.5 38 1 15.5 25 29.5 2928 30.5 31.5 26.5 35.5 19 2 24 26 30.5 34 26.5 31 35 28 38.5 38 4 27 2831 36 32 29.5 35.5 28 36 42 6 27 26 32 31.5 34 31 33.5 31 49 38.5 8 32.526.5 31.5 35.5 31 39.5 30.5 29.5 40 38 12 31 29 37.5 38.5 37 30 18 3134.5 33 24 28.5 34 41 34 42 39.5 30.5 32.5 38 47 48 28 34 31 39.5 42 2930.5 27.5 40.5 27.5 72 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a 96 n/an/a n/a n/a n/a n/a n/a n/a n/a n/a 168 n/a 31 28 n/a 30 30.5 n/a 32 n/a31 336 n/a 31.5 n/a n/a 29 n/a n/a 33.5 n/a 40

MOD-5014 Characterization in Hemophilic Dogs by Whole BloodKaolin-Thromboelastography (WB Kaolin-TEG)

Kaolin-TEG parameters were monitored at designated time points postMOD-5014 administration for all dogs and at all doses. The objectives ofthis specific analysis were to characterize the kinetics of TEG withtime, to assess the minimal dose in which normalization of the differentparameters (R, K, Angle, Mass.) occurred across different animals, thedose-dependent response within individuals and to finally compare theoverall TEG profile following MOD-5014 administration to published datafor rhFVIIa at a similar dose range as well as to normal values obtainedin healthy dogs (Knudsen et al., 2011). The individual data is providedin FIGS. 101 (A-D), 102 (A-D), 103 (A-D), 104 (A-D), 105 (A-D) and 106(A-D).

MOD-5014 Whole Blood Kaolin-TEG Performance Following Administration atDifferent Doses

The initial dose of 50 μg/kg administered to 2 dogs was unable tosignificantly improve TEG performance, as reflected by poor R, K, angleand MA values post-administration (animal P14; FIGS. 102 (A-D)), but themajority of the parameters were improved when the dose was increased to200 μg/kg. In the second animal (N06) TEG values were also not improvedfollowing administration of 50 μg/kg MOD-5014; this might be aconsequence of the partially normalized TEG values at pre-dose, whichpotentially had a direct effect on the poor post-administrationresponse, although no further improvement was observed post-dosing.Based on the above it was proposed that the 50 μg/kg dose is notadequate to support and correct the hemostatic defect in the testeddogs.

Administration of 200 μg/kg MOD-5014 in the majority of the animalscorrected the TEG parameters to values which are still below normalrange, yet provided an effect which is significantly more pronouncedthan the 50 μg/kg dose. At a dose of 400 μg/kg a slight improvement inTEG values and a prolonged response was obtained (FIGS. 102 (A-D); 103(A-D) and 104 (A-D)). When dosing two different animals with 600 μg/kg,the response in one animal (Joanie; FIG. 105 (A-D)) was comparable toanimals dosed with 400 μg/kg. While the K time, Angle and MA values inanimal N05 (FIG. 106 (A-D)) reached the normal range, this might also bea consequence of improved pre-dose values and inter-animal variability.It appears that administration of MOD-5014 at a range of doses enabledpartial correction of TEG values, while the optimal minimal effectivedose was at the range of 200-400 μg/kg of MOD-5014.

Intra-Animal Variability of MOD-5014 Whole Blood Kaolin-TEGDose-Dependent Response

An intra-animal dose-dependent improvement was observed when a higherdose was injected to the same animal. Some variation at different timepoints was observed, that could reflect normal biological variation(FIGS. 101 (A-D), 102 (A-D), 103 (A-D), 104 (A-D), 105 (A-D) and 106(A-D)). The improvement was most pronounced when dosing at 200 and 400μg/kg (Blondie and Josie), as presented in FIGS. 101 (A-D), 102 (A-D),103 (A-D), 104 (A-D), 105 (A-D) and 106 (A-D).

Reproducibility of MOD-5014 Whole Blood Kaolin-TEG Results BetweenIndividuals (Inter-Animal Variability)

As observed in FIGS. 101 (A-D), 102 (A-D), 103 (A-D), 104 (A-D), 105(A-D) and 106 (A-D), although animals corrected TEG values followingadministration of MOD-5014, they demonstrated different profiles throughthe time of measurement. This might be a result of biologicalvariability, considering the different genetic background of each animaland the slight differences in pre-dose values. The observed variationactually mimics the clinical setting, in which individual TEG values mayvary between individuals receiving similar dosing, and may eventuallytranslate to different efficacy and bleeding control capability ofFVIIa.

MOD-5014 as a Long-Acting Product

Comparison of the correction capability of MOD-5014 and rhFVIIa suggeststhat MOD-5014 maximum and minimum values are very similar to the resultsreported for hFVIIa. However, a marked improvement in the duration ofthe reaction was observed with MOD-5014. Following hFVIIa administrationthe majority of the hFVIIa analyzed parameters dropped back to baseline4 hours post-dosing (FIGS. 107(A-D)), while MOD-5014 enables an extendedeffect reaching baseline values 24 hours post-dosing and provides a moresustained and prolonged response (FIGS. 108(A-D); marked with blackarrows). The data suggests that the duration of the TEG responsecorrelates to FVIIa short PK-PD profile in hemophilic dogs, which wasfurther investigated, subsequently confirming the extended responsefollowing MOD-5014 administration.

Activated Partial Thromboplastin Time (aPTT) Performance

Using the Triniclot reagent as activator and a 60 second incubation, theaPTT values for canine hemophilia A plasma were 65.6 seconds (data notshown). Following MOD-5014 administration, aPTT was reduced in adose-dependent manner down to 40 sec. When comparing this information topreviously reported data, it was found to be very similar to the valuesobtained pre- and post-administration of 193 μg/kg rhFVIIa to aFVIII-deficient dog (Brinkhous et al., 1989), while a retrospectiveanalysis suggests a prolonged effect when administrating 200 μg/kgMOD-5014, with a subsequent return to baseline aPTT values 24 hourspost-dosing.

Conclusions:

In this study, the safety, PK, PD and hemophilic coagulopathy correctionability of a novel, long-acting FVIIa (MOD-5014) was assessed inFVIII-deficient dogs. MOD-5014 was administered in relevant clinicaldoses, while the potential minimal effective dose in the dogs wasinitially established based on an ex vivo study.

MOD-5014 was well tolerated by all dogs and no adverse events wereobserved. Dose-dependent response was observed in both the PK and PD(activation measurement) analyses, further confirming the long-actingproperties of MOD-5014 in comparison to published parameters (Knudsen etal. 2011).

Recombinant human FVIIa has been shown previously to be efficacious intreating hemophilic canine bleeds. A marked improvement of TEGcoagulation profile was observed following administration of MOD-5014 atdoses of 200-400 μg/kg, while a lower dose of 50 μg/kg was unable tocorrect TEG values, suggesting that it is below the minimal effectivedose in dogs. A dose-dependent response was observed following MOD-5014administration, while inter-individual variation was higher thanexpected and might be a consequence of biological variability. Whencomparing MOD-5014 performance to published data, MOD-5014 had apronounced and prolonged effect at lower doses (270 and 200 μg/kg,respectively). MOD-5014 in vivo activity was also reflected by areduction in aPTT values, further confirming a sustained activation ofthe coagulation system.

In general, testing MOD-5014 in hemophilic dogs offers severaladvantages over rodent models such as mice and rats. Hemophilic dogsexhibit a disease phenotype closely recapitulating that of humans;moreover, dogs are more comparable with humans in regard to body weightas well as to FVIIa dosing requirements and to pharmacokineticcharacteristics.

Overall, this study further confirmed the longevity of MOD-5014 in arelevant, well-established model previously shown to provide an accuraterecapitulation of human biology, specifically with respect tohemostasis. The data obtained in this study have significant value andprovide the first evidence that MOD-5014 is a safe and effectivelong-acting FVIIa in large animals that can be used potentially as anagent for both prophylactic and on-demand treatment of hemophilicpatients with inhibitors. Thus, MOD-5014 has a tremendous potential tosignificantly benefit patients by reducing the frequency ofadministration and enabling prophylactic use.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

What is claimed is:
 1. A CTP-modified polypeptide consisting of acoagulation factor and three chorionic gonadotropin carboxy terminalpeptides (CTPs) attached to the carboxy terminus of said coagulationfactor.
 2. The CTP-modified polypeptide of claim 1, wherein the sequenceof at least one CTP is selected from the group consisting of: SEQ ID NO:1 and SEQ ID NO:
 2. 3. The CTP-modified polypeptide of claim 1, whereinat least one CTP is glycosylated.
 4. The CTP-modified polypeptide ofclaim 1, wherein at least one CTP is truncated.
 5. The CTP-modifiedpolypeptide of claim 1, wherein at least one CTP is attached to saidcoagulation factor via a linker
 6. The CTP-modified polypeptide of claim5, wherein said linker is a peptide bond.
 7. The CTP-modifiedpolypeptide of claim 1, wherein said coagulation factor is a vitamin Kdependent glycoprotein.
 8. The CTP-modified polypeptide of claim 1,wherein said coagulation factor is Factor IX.
 9. The CTP-modifiedpolypeptide of claim 1, wherein said coagulation factor is Factor VII.10. The CTP-modified polypeptide of claim 1, wherein said coagulationfactor is activated FVII (FVIIa).
 11. A pharmaceutical compositioncomprising the CTP-modified polypeptide of claim 1 and apharmaceutically acceptable carrier.
 12. A polynucleotide moleculecomprising a coding portion encoding a CTP-modified polypeptideconsisting of a coagulation factor and three chorionic gonadotropincarboxy terminal peptides (CTPs) attached to the carboxy terminus ofsaid coagulation factor.
 13. The polynucleotide molecule of claim 12,wherein the sequence of at least one CTP is selected from the groupconsisting of: SEQ ID NO: 1 and SEQ ID NO:
 2. 14. The polynucleotidemolecule of claim 12, wherein at least one CTP is truncated.
 15. Thepolynucleotide molecule of claim 12, wherein said coagulation factor isa vitamin K dependent glycoprotein.
 16. The polynucleotide molecule ofclaim 12, wherein said coagulation factor is Factor IX.
 17. Thepolynucleotide molecule of claim 12, wherein said coagulation factor isFactor VII.
 18. The polynucleotide molecule of claim 12, wherein saidcoagulation factor is activated FVII (FVIIa).
 19. An expression vectorcomprising the polynucleotide molecule of claim
 12. 20. A cellcomprising the expression vector of claim
 20. 21. A compositioncomprising the expression vector of claim
 20. 22. A method of extendingthe biological half-life of a coagulation factor, comprising the step ofattaching three chorionic gonadotropin carboxy terminal peptides to thecarboxy terminus of said coagulation factor, thereby extending thebiological half-life of said coagulation factor.
 23. The method of claim22, wherein at least one CTP is encoded by an amino acid sequenceselected from the group consisting of: SEQ ID NO:1 and SEQ ID NO:
 2. 24.The method of claim 22, wherein at least one CTP is glycosylated. 25.The method of claim 22, wherein at least one CTP is truncated.
 26. Themethod of claim 22, wherein at least one CTP is attached to saidcoagulation factor via a linker.
 27. The method of claim 26, whereinsaid linker is a peptide bond.
 28. A method of improving the area underthe curve (AUC) of a coagulation factor, comprising the step ofattaching three chorionic gonadotropin carboxy terminal peptides to thecarboxy terminus of said coagulation factor, thereby improving the AUCof said coagulation factor.
 29. The method of claim 28, wherein at leastone CTP is encoded by an amino acid sequence selected from the groupconsisting of: SEQ ID NO:1 and SEQ ID NO:
 2. 30. The method of claim 28,wherein at least one CTP is glycosylated.
 31. The method of claim 28,wherein at least one CTP is truncated.
 32. The method of claim 28,wherein at least one CTP is attached to said coagulation factor via alinker.
 33. The method of claim 32, wherein said linker is a peptidebond.
 34. A method of reducing the dosing frequency of a coagulationfactor, comprising the step of attaching three chorionic gonadotropincarboxy terminal peptides to the carboxy terminus of said coagulationfactor, thereby reducing the dosing frequency of said coagulationfactor.
 35. The method of claim 34, wherein at least one CTP is encodedby an amino acid sequence selected from the group consisting of: SEQ IDNO:1 and SEQ ID NO:
 2. 36. The method of claim 34, wherein at least oneCTP is glycosylated.
 37. The method of claim 34, wherein at least oneCTP is truncated.
 38. The method of claim 34, wherein at least one CTPis attached to said coagulation factor via a linker.
 39. The method ofclaim 38, wherein said linker is a peptide bond.
 40. A method ofpreventing or treating a blood clotting or coagulation disorder in asubject, the method comprising the step of administering a CTP-modifiedcoagulation factor polypeptide of claim 1 to said subject, therebypreventing or treating a blood clotting or coagulation disorder in saidsubject.
 41. The method of claim 40, wherein said disorder ishemophilia.
 42. The method of claim 40, wherein said subject is a humanchild.
 43. The method of claim 40, wherein said administering is via thesubcutaneous route.